Long-acting formulations

ABSTRACT

This invention concerns pharmaceutical compositions for administration via intramuscular or subcutaneous injection, comprising micro- or nanoparticles of the anti-TB compound bedaquiline, suspended in an aqueous pharmaceutically acceptable carrier, and the use of such pharmaceutical compositions in the treatment and prophylaxis of a pathogenic mycobacterial infection.

FIELD OF THE INVENTION

This invention concerns pharmaceutical compositions for administrationvia intramuscular or subcutaneous injection, comprising micro- ornanoparticles of the ATP synthase inhibitor compound, bedaquiline(marketed as Sirturo®, where bedaquiline is in the form of its fumaratesalt), suspended in an aqueous pharmaceutically acceptable carrier, andthe use of such pharmaceutical compositions in the treatment ofbacterial infections, e.g. tuberculosis and the like.

BACKGROUND OF THE INVENTION

Bedaquiline is a known anti-tuberculosis drug used in variouscombinations. It may be formulated in the form of a pharmaceuticallyacceptable salt, such as in the form of bedaquiline fumarate, marketedas Sirturo®. It is thought to act as an ATP synthase inhibitor,possessing a selectivity index of more than 20000 for mycobacterial ATPsynthase versus eukaryotic mitochondrial ATP synthase.

Bedaquiline has already been reported as being useful in the treatmentof mycobacterial infections, as well as being useful in killing dormant,latent, persistent mycobacteria, in particular Mycobacteriumtuberculosis, and can consequently be used to treat latent TB.

Such use of bedaquiline has been described in several publicationsincluding international patent documents WO 2004/011436 and WO2006/067048. It is also known that bedaquiline is bactericidal againstmycobacterium leprae, for example as described in “Bacterial Activitiesof R207910 and other Antimicrobial Agents against Mycobacterium lepraein Mice”, Antimicrobial agents and Chemotherapy, April 2006, p 1558, and“The Diarylquinolone R207910 is Bactericidal against Mycobacteriumleprae in mice and at Low Dose Administered Intermittently”,Antimicrobial agents and Chemotherapy, September 2009, p 3989.

The goal of long-acting formulations can be to reduce drug burden. Thisis particularly useful for treatment regimens that may last severalmonths.

The number and/or volume of dosage forms that need to be administeredare commonly referred to as “pill burden”. A high pill burden isundesirable for many reasons, such as the frequency of intake, oftencombined with the inconvenience of having to swallow large dosage forms,as well as the need to store and transport a large number or volume ofpills. A high pill burden increases the risk of patients not takingtheir entire dose, thereby failing to comply with the prescribed dosageregimen. As well as reducing the effectiveness of the treatment, thismay also lead to the emergence of resistance (e.g. in the case ofbedaquiline, bacterial resistance).

It would be attractive to provide therapy involving the administrationof dosage forms at long time intervals such as one week or longer, oreven one month or longer.

Various formulations are known in the art, including long-acting ones.For instance, micro- and nano-suspension technology is known forachieving long-acting formulations in the field of anti-HIV drugs, forinstance as described in international patent applications WO2007/147882 and WO 2012/140220. Further, nanoparticles known in theprior art have been described, for example, in EP-A-0 499 299. Suchparticles have an average particle size in the submicron range andconsist of particles of a crystalline drug substance having a surfacemodifier adsorbed on their surface. Nanoparticles have also been used toformulate poorly water-soluble active ingredients.

Long-acting formulations of the anti-tuberculosis drug bedaquiline arenow described. It now has been found that the compound bedaquiline canbe formulated into micro- or nanoparticles and that such formulationscan be used as long-acting (or depot) formulations, which may find usein the treatment of various bacterial infections, including e.g.tuberculosis.

Challenges for such formulations would have been thought to exist basedon pharmacokinetic (PK) properties of tuberculosis drugs, includingbedaquiline, and the need to keep plasma levels above a minimum levelbearing those PK properties in mind. The mean terminal eliminationhalf-life of bedaquiline and the N-monodesmethyl metabolite (also knownas the M2 metabolite) is approximately 5.5 months. This long terminalelimination phase likely reflects slow release of bedaquiline and M2from peripheral tissues. In October 2016 at the UNION conference inLiverpool Susan Swindells from the University of Nebraska Medical Centerpresented on “Experience from Long-Acting HIV Drug Development” where itwas summarsied that existing tuberculosis drugs were not idealcandidates (for long-acting) and reliable pharmacodynamic models werelacking.

The invention furthermore relates to the intermittent administration ofthese micro- or nanoparticle formulations at time intervals of one weekor longer that result in plasma levels that may be sufficient tosuppress the growth of the mycobacterial infection. This allows for areduced number of administrations thereby being beneficial in terms ofpill burden and drug compliance of the patient. The micro- ornanoparticle formulations of bedaquiline of the invention therefore maybe useful in the long-term treatment of mycobacterial infections (e.g.tuberculosis, including latent tuberculosis, and leprosy).

The intermittent administration of micro- or nanoparticle formulationsof bedaquiline at time intervals of one week or longer furthermoreresults in plasma levels that may be sufficient to provide preventionagainst transmission of mycobacterial infection. Also in this instance,a reduced number of administrations is required, which again isadvantageous in terms of pill burden and drug compliance of theindividual at risk of being infected.

SUMMARY OF THE INVENTION

The present invention is concerned with a pharmaceutical composition foradministration by intramuscular or subcutaneous injection, comprising atherapeutically effective amount of bedaquiline, or a pharmaceuticallyacceptable salt thereof, in the form of a suspension of micro- ornanoparticles comprising:

-   (a) bedaquiline, or a pharmaceutically acceptable salt thereof, in    micro- or nanoparticle form, and a surface modifier; and-   (b) a pharmaceutically acceptable aqueous carrier,    wherein such a composition may be referred to herein as    “composition(s) of the invention”.

The composition of the invention is a suspension, by which we mean thatthe bedaquiline active ingredient is suspended in the pharmaceuticallyacceptable aqueous carrier.

The composition of the invention (i.e. the suspension) contains asurface modifier, which may be adsorbed onto the surface of the activeingredient bedaquiline.

In an embodiment, the present invention may therefore concern apharmaceutical composition for administration by intramuscular orsubcutaneous injection, comprising a therapeutically effective amount ofbedaquiline, or a pharmaceutically acceptable salt thereof, in the formof a suspension of micro- or nanoparticles comprising:

-   (a) bedaquiline, or a pharmaceutically acceptable salt thereof, in    micro- or nanoparticle form, having a surface modifier adsorbed to    the surface thereof; and-   (b) a pharmaceutically acceptable aqueous carrier; wherein the    bedaquiline active ingredient is suspended.

The invention further concerns a method of treating a subject infectedwith pathogenic mycobacteria such as Mycobacterium tuberculosis, M.bovis, M. leprae, M. avium and M. marinum. In an embodiment, themycobacteria is Mycobacterium tuberculosis (including the latent ordormant form) or Mycobacterium leprae. The compositions of the inventionmay be particularly suitable for the treatment of Mycobacterium lepraeand the latent or dormant form of Mycobacterium tuberculosis. This isbecause for treating these specific infections, a lower concentration ofbedaquiline in the plasma may be effective against such infection, forinstance as described in Antimicrobial Agents and Chemotherapy,September 2009, p. 3989-3991 by Robert Gelber, Koen Andries et al (thecontents of which are hereby incorporated by reference, and wherein,essentially, it is reported that low and intermittent dosing withbedaquiline holds promise for leprosy patients; whereas minimal dosekilling 99% of bacilli for M. tuberculosis is 30 mg/kg/wk, for M. leprait is <5.0 mg/kg/wk, and hence dosing once a month may be as efficientas 5 days a week; other publications of the effect of bedaquiline onMycobacterium leprae in mice include Antimicrobial Agents andChemotherapy, April 2006, p. 1558-1560 by Baohong Ji, Koen Andries etal—the contents of which are also hereby incorporated by reference).Hence, the compositions of the invention may be particularly suitable ina method of treating a subject infected with Mycobacterium leprae or thelatent/dormant form of Mycobacterium tuberculosis. Such methods oftreating a subject infected with pathogenic mycobacteria comprise theadministration, by intramuscular or subcutaneous injection, of atherapeutically effective amount of a pharmaceutical composition asspecified above or hereinafter. Or, alternatively, the inventionconcerns the use of a pharmaceutical composition as specified above orhereinafter, for the manufacture of a medicament for treating pathogenicmycobacteria infection (or for using such medicament in a particulartreatment regime as described herein). In one embodiment, thecomposition is for the long-term treatment of pathogenic mycobacteriainfection. In an embodiment, the pathogenic mycobacterial infection maysuch as described above or hereinafter, such as an infection thatrequires long-term treatment (in a further embodiment, an infection thatfurther may be treated at relatively low plasma concentration levels ofbedaquiline or its active metabolite, for instance latent/dormantMycobacterium tuberculosis or, in a particular embodiment, Mycobacteriumleprae).

In another aspect, there is provided a method for the long termtreatment of a subject infected with pathogenic mycobacteria such asMycobacterium tuberculosis, M. bovis, M. leprae, M. avium and M.marinum, said method comprising the administration of an effectiveamount of a pharmaceutical composition as specified above orhereinafter, for administration by intramuscular or subcutaneousinjection; wherein the composition is administered or is to beadministered intermittently at a time interval that is in the range ofone week to one year, or one week to two years. Or, alternatively, theinvention concerns the use of a pharmaceutical composition as specifiedabove or hereinafter, for the manufacture of a medicament for the longterm treatment of a subject infected with pathogenic mycobacteria suchas Mycobacterium tuberculosis, M. bovis, M. leprae, M. avium and M.marinum, for administration by intramuscular or subcutaneous injection,wherein the composition is administered or is to be administeredintermittently at a time interval that is in the range of one week toone year, or one week to two years. Hence, it will be understood thatthe term “long term treatment” refers to treatment where one dose or oneadministration (e.g. by intramuscular or subcutaneous injection) willhave a persistent therapeutic effect over a time period, as describedherein, for instance a persistent therapeutic effect over several hours,weeks or months (e.g. in an embodiment, over a period of at least or upto one month, three months or six months); see examples. Put anotherway, long term treatment may refer to, where there is more than onedose/administration, the long period of time (as described herein)between the doses/administrations, i.e. the intervals are a long periodof time as described herein.

In another aspect, there is provided a method for the long termtreatment of a subject infected with pathogenic mycobacteria (e.g. ofany of the types as described here), as described herein (e.g. above)wherein one dose or administration (e.g. of the amount described herein,e.g. hereinafter) is provided/required (and has a persistent effect,e.g. over a time period described herein). In another aspect, there isprovided such a long term treatment regime, where two such doses oradministrations are provided/required, which doses/administrations aregiven at intervals, wherein the interval time period is that asdescribed herein, e.g. a period of at least or up to one month, threemonths or six months—for instance for a period of time in whichpersistent therapeutic effect lasts). In a further embodiment, there isprovided such a long term treatment regime, in which three such doses oradministrations are provided/required at such intervals as hereindescribed. In yet a further embodiment, there is provided a long termtreatment regime as herein described but which is preceded with alead-in treatment phase (that is not a long term treatment regime, e.g.a once-daily administration course, lasting for one week, two weeks,three weeks or one month).

The invention further concerns a method for the prevention of apathogenic mycobacterial infection in a subject at risk of beinginfected by a pathogenic mycobacterial infection, said method comprisingadministering an amount, effective in preventing a pathogenicmycobacterial infection, of a pharmaceutical composition as specifiedabove or as further specified hereinafter, to said subject. Oralternatively, the invention concerns the use of a pharmaceuticalcomposition as specified above or as further specified hereinafter forthe manufacture of a medicament for the prevention of a pathogenicmycobacterial infection in a subject at risk of being infected by apathogenic mycobacterial infection.

In another aspect the invention relates to a method for the long termprevention of a pathogenic mycobacterial infection in a subject at riskof being infected by a pathogenic mycobacterial infection, said methodcomprising administering to said subject an effective amount of apharmaceutical composition as specified above or as further specifiedhereinafter, wherein the composition is administered or is to beadministered intermittently at a time interval that is in the range ofone week to one year, or one week to two years.

The present invention furthermore relates to the use of a pharmaceuticalcomposition as specified above or as further specified hereinafter, forthe manufacture of a medicament for the long term prevention for thelong term prevention of a pathogenic mycobacterial infection in asubject at risk of being infected by a pathogenic mycobacterialinfection, wherein the composition is administered or is to beadministered intermittently at a time interval that is in the range ofone week to one year or one week to two years.

In one embodiment the invention concerns a use or a method as specifiedherein, wherein the pharmaceutical composition is administered or is tobe administered at a time interval that is in the range of one week toone month, or in the range of one month to three months, or in the rangeof three months to six months, or in the range of six months to twelvemonths, or in the range of 12 months to 24 months.

In another embodiment the invention concerns a use or a method asspecified herein, wherein the pharmaceutical composition is administeredor is to be administered once every two weeks, or once every month, oronce every three months.

Further pharmaceutical compositions, methods of treatment or prevention,as well as uses for the manufacture of medicaments based on thesecompositions will be described hereinafter and are meant to be part ofthe present invention.

The invention is also described with reference to the following figures:

FIG. 1: “Plasma kinetics of TMC207 (bedaquiline; BDQ) and M2(bedaquiline's metabolite; see herein) in mouse, after a single dose of30 mg/kg”

FIG. 2: “Plasma kinetics of TMC207 in mouse when administered IM or SCwith 200 mg/ml formulations (specifically formulations of Examples 1Aand 1B, i.e. the nano- and micro-suspension, respectively) at a dose of160 mg/kg” (TMC207 is referred to the in the Figure as “UD”)

FIG. 3: “Plasma kinetics of M2 in mouse when administered IM or SC with200 mg/ml formulations (specifically formulations of Examples 1A and 1B,i.e. the nano- and micro-suspension, respectively) at a dose of 160mg/kg” (M2 is referred to in the Figure as “met”)

FIG. 4: “Plasma kinetics of TMC207 in mouse when administered IM or SCwith 100 mg/ml formulations (specifically formulations of Examples 1Cand 1D, i.e. the nano- and micro-suspension, respectively) at a dose of80 mg/kg” (TMC207 is referred to the in the Figure as “UD”)

FIG. 5: “Plasma kinetics of M2 in mouse when administered IM or SC with100 mg/ml formulations (specifically formulations of Examples 1C and 1D,i.e. the nano- and micro-suspension, respectively) at a dose of 80mg/kg” (M2 is referred to in the Figure as “met”)

FIG. 6: “Plasma kinetics of TMC207 in male rats when administered IM orSC with 200 mg/ml micro-formulation (see Example 1, Formulation 1B i.e.the micro-suspension) at a dose of 40 mg/kg” and “Plasma kinetics ofTMC207 in male rats when administered IM or SC with 200 mg/mlnano-formulation (see Example 1, Formulation 1A, i.e. thenano-suspension) at a dose of 40 mg/kg”

FIG. 7: “Plasma kinetics of TMC207 in male beagle dogs when administeredIM or SC with 200 mg/ml micro-formulation (see Example 1, Formulation1B) at a dose of 40 mg/kg” and “Plasma kinetics of TMC207 in male beagledogs when administered IM or SC with 200 mg/ml nano-formulation (seeExample 1, Formulation 1A) at a dose of 40 mg/kg”

DETAILED DESCRIPTION OF THE INVENTION

The compound used in the invention is the compound TMC207, also referredto as bedaquiline.

Bedaquiline can be used in its non-salt form or as a suitablepharmaceutically acceptable salt form, such as an acid addition saltform or base addition salt form. In an embodiment, bedaquiline is in itsnon-salt form in compositions of the invention.

The pharmaceutically acceptable acid addition salts are defined tocomprise the therapeutically active non-toxic acid addition salt formswhich bedaquiline is able to form. Said acid addition salts can beobtained by treating the free form of bedaquiline with appropriateacids, for example inorganic acids, for example hydrohalic acid, inparticular hydrochloric acid, hydrobromic acid, sulfuric acid, nitricacid and phosphoric acid; organic acids, for example acetic acid,hydroxyacetic acid, propanoic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid, malicacid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonicacid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid,salicyclic acid, p-aminosalicylic acid and pamoic acid. In particular,the fumarate salt is considered, given that this is the form employed inthe already-marketed product Sirturo®.

Possible therapeutically active non-toxic base addition salt forms maybe prepared by treatment with appropriate organic and inorganic bases.Appropriate base salts forms comprise, for example, the ammonium salts,the alkaline and earth alkaline metal salts, in particular lithium,sodium, potassium, magnesium and calcium salts, salts with organicbases, e.g. the benzathine, N-methyl-D-glucamine, hybramine salts, andsalts with amino acids, for example arginine and lysine.

Conversely, said acid or base addition salt forms can be converted intothe free forms by treatment with an appropriate base or acid.

The term addition salt as used in the framework of this application alsocomprises the solvates which bedaquiline as well as the salts thereof,are able to form. Such solvates are, for example, hydrates andalcoholates.

Whenever reference to bedaquiline (or TMC207) is employed herein, werefer to the single stereoisomeric form that is employed in the marketedproduct Sirturo®, and which is disclosed in WO2004/011436 as anantimycobacterial agent.

It has been found that the physico-chemical properties of bedaquilineallow for the manufacture of micro- or nanoparticle suspensions thathave unique pharmacokinetic properties in that they can be used for thelong term treatment of a pathogenic mycobacterial infection as well asin the long term prevention of a pathogenic mycobacterial infection andto this purpose only a limited number of drug administrations isrequired. This is beneficial in terms of pill-burden as well as patientcompliance with the prescribed dose regimen.

As used herein the term “treatment of a pathogenic mycobacterialinfection” relates to the treatment of a subject being infected with apathogenic mycobacterial infection.

The term “prevention of a pathogenic mycobacterial infection” relates tothe prevention or avoidance of a subject becoming infected with apathogenic mycobacterial infection. The source of infection can bevarious, for instance a material containing a pathogenic mycobacterialinfection.

The terms “therapeutically effective amount”, “an amount, effective inpreventing a pathogenic mycobacterial infection”, and similar terms,refer to amounts, or concentrations, of the compositions of theinvention (or amounts/concentrations of active ingredient bedaquilinewithin such compositions) that result in efficacious plasma levels. With“efficacious plasma levels” it is meant those plasma levels ofbedaquiline that provide effective treatment or effective prevention ofa pathogenic mycobacterial infection. This is becauseamount/dose/administration given may be linked to the desired exposurelevels or desired plasma levels for the effective treatment/prevention,for instance as described herein (see e.g. the examples).

The term “subject” in particular relates to a human being.

The term “micro- or nanoparticles” refers to particles in the micrometeror nanometer range. The size of the particles should be below a maximumsize above which administration by subcutaneous or intramuscularinjection becomes impaired or is even no longer possible. Said maximumsize depends for example on the limitations imposed by the needlediameter or by adverse reactions of the body to large particles, orboth. In one embodiment, the pharmaceutical compositions of theinvention comprise bedaquiline in microparticle form. In anotherembodiment, the pharmaceutical compositions of the invention comprisebedaquiline in nanoparticle form. The average effective particle size ofthe micro- or nanoparticles of the present invention may be below about50 μm, or below about 20 μm, or below about 10 μm, or below about 1000nm, or below about 500 nm, or below about 400 nm, or below about 300 nm,or below about 200 nm. The lower limit of the average effective particlesize may be low, e.g. as low as about 100 nm or as low as about 50 nm.In one embodiment, the average effective particle size is in the rangeof about 50 nm to about 50 μm, or about 50 nm to about 20 μm, or about50 nm to about 10 μm, or about 50 nm to about 1000 nm, about 50 nm toabout 500 nm, or about 50 nm to about 400 nm, or about 50 nm to about300 nm, or about 50 nm to about 250 nm, or about 100 nm to about 250 nm,or about 150 nm to about 220 nm, or 100 to 200 nm, or about 150 nm toabout 200 nm, e.g. about 130 nm, or about 150 nm. For instance, bothafter preparation and after a period of time of up to 3 months (e.g.when stored at temperatures of about 5° C., 25° C. and 40° C.)generally:

-   -   the micro-suspensions may have, in an embodiment, a D90 of        between about 3 and 10 μm (e.g. about 3.5, 4 or 5 μm) and a D50        of between about 2 and 4 μm (e.g. about 3 μm)    -   the nano-suspensions may have, in an embodiment, a D90 of        between about 0.5 and 1.5 μm (e.g. about, or less than 1 μm or        about, or less than about 1000 nm) and a D50 of between about        0.1 and 0.5 μm (e.g. about, or less than, about 0.3 μm, or less        than about 300 nm).

In an embodiment, the micro-particles are employed, wherein the averageeffective particle size, as measured by D10, D50 and/or D90 (in anembodiment as measured by D50) is below about 50 μm, or below about 20μm, and above about 0.1 μm (100 nm). In an embodiment the range for suchmicro-particles employed in the compositions of the invention is betweenabout 20 μm and about 0.1 μm (in a further embodiment between about 15μm, and above about 0.2 μm (200 nm) and in a further embodiment betweenabout 10 μm, and above 0.5 μm (500 nm), for instance between about 10μm, and above 1 μm or below about 1000 nm, or below about 500 nm, orbelow about 400 nm, or below about 300 nm, or below about 200 nm. Theforegoing values are refer to measurements after preparation. They mayalso, however, in an embodiment, refer to measurements after a period oftime up to 3 months (e.g. after 5 days, one week, two weeks, one month,two months or three months) and stored at various temperatures (e.g. attemperatures of about 5° C., 25° C. and 40° C.).

As used herein, the term average effective particle size has itsconventional meaning as known to the person skilled in the art and canbe measured by art-known particle size measuring techniques such as, forexample, sedimentation field flow fractionation, photon correlationspectroscopy, laser diffraction or disk centrifugation. The averageeffective particle sizes mentioned herein may be related to volumedistributions of the particles. In that instance, by “an effectiveaverage particle size of less than about 50 μm” it is meant that atleast 50% of the volume of the particles has a particle size of lessthan the effective average of 50 μm, and the same applies to the othereffective particle sizes mentioned. In a similar manner, the averageeffective particle sizes may be related to weight distributions of theparticles but usually this will result in the same or about the samevalue for the average effective particle size.

The pharmaceutical compositions of the present invention provide releaseof the active ingredient bedaquiline over a prolonged period of time andtherefore they can also be referred to as sustained or delayed releasecompositions. After administration, the compositions of the inventionstay in the body and steadily release bedaquiline, keeping such levelsof this active ingredient in the patient's system for a prolonged periodof time, thereby providing, during said period, the appropriatetreatment or prevention of a pathogenic mycobacterial infection. Becauseof the fact that the pharmaceutical compositions of the invention stayin the body and steadily release bedaquiline (and its active metabolite,referred to as M2 herein; see hereinafter, the methyl-substitutedmetabolite), they can be referred to as pharmaceutical compositionssuitable as long-acting (or depot) formulations.

As used herein with the term “prolonged period of time”, there is meanta term (or time period) that may be in the range of one week up to oneyear or up to two years, or a term in the range of one to two weeks, ortwo to three weeks, or three to four weeks, or a term in the range ofone to two months, or two to three months, or three to four months, orthree to six months, or six months to 12 months, or 12 months to 24months, or a term that is in the range of several days, e.g. 7, 10 or 12days, or several weeks, e.g. 2, 3 or 4 weeks, or one month, or severalmonths, e.g. 2, 3, 4, 5 or six months or even longer, e.g. 7, 8, 9 or 12months.

The pharmaceutical compositions of this invention may be applied in thelong-term treatment or the long-term prevention of a pathogenicmycobacterial infection, or with other words they may be used in thetreatment of a pathogenic mycobacterial infection, or in the preventionof a pathogenic mycobacterial infection, during a prolonged period oftime. The compositions of the invention are effective in the treatmentor prevention of a pathogenic mycobacterial infection for a prolongedperiod of time, for example for at least about one week or longer, orfor about 1 month or longer. By the expression “effective for at leastabout one week or longer”, one means that the plasma level of the activeingredient, bedaquiline (and/or its active metabolite M2), should beabove a threshold value. In case of therapeutic application saidthreshold value is the lowest plasma level at which bedaquiline (and/orits active metabolite M2) provides effective treatment of a pathogenicmycobacterial infection. In case of application in the prevention of apathogenic mycobacterial infection said threshold value is the lowestplasma level at which bedaquiline (and/or its active metabolite M2) iseffective in preventing transmission of a pathogenic mycobacterialinfection.

With “long term” for example as used in relation to “long termprevention of a pathogenic mycobacterial infection” or “long termtreatment of a pathogenic mycobacterial infection”, or similarterminology, there are meant terms that may be in the range of one weekup to one year or up to two years, or longer, such as five or 10 years.In particular in the case of treatment of a pathogenic mycobacterialinfection, such terms will be long, in the order of one to severalmonths, one year or longer. Such terms may also be relatively short, inparticular in the case of prevention. Shorter terms are those of severaldays, e.g. 7, 10 or 12 days, or several weeks, e.g. 2, 3 or 4 weeks, orone month, or several months, e.g. 2, 3, 4, 5 or six months or evenlonger, e.g. 7, 8, 9 or 12 months. In one embodiment the methods anduses in accordance with the present invention are for the prevention ofa pathogenic mycobacterial infection during one month, or severalmonths, e.g. 2, 3, 4, 5 or six months or even longer, e.g. 7, 8, 9 or 12months.

The pharmaceutical compositions of the present invention can beadministered at various time intervals. When used in the prevention of apathogenic mycobacterial infection, the pharmaceutical compositions ofthis invention can be administered only once or a limited number oftimes such as twice, three, four, five or six times, or more. This maybe recommendable where prevention is required during a limited period oftime, such as the period during which there is a risk of infection.

The pharmaceutical compositions of the present invention can beadministered at the time intervals mentioned above, such as at a timeinterval that is in the range of one week to one month, or in the rangeof one month to three months, or in the range of three months to sixmonths, or in the range of six months to twelve months. In oneembodiment, the pharmaceutical composition can be administered onceevery two weeks, or once every month, or once every three months. Inanother embodiment the time interval is in the range of one to twoweeks, or two to three weeks, or three to four weeks, or the timeinterval is in the range of one to two months, or two to three months,or three to four months, or three to six months, or six months to 12months, or 12 months to 24 months. The time interval may be at least oneweek, but may also be several weeks, e.g. 2, 3, 4, 5 or 6 weeks, or attime intervals of one month, or of several months, e.g. 2, 3, 4, 5 or 6months or even longer, e.g. 7, 8, 9 or 12 months. In one embodiment, thepharmaceutical compositions of the present invention are administered ata time interval of one, two or three months. These longer periodsbetween each administration of the pharmaceutical compositions of theinvention provide further improvements in terms of pill burden andcompliance. To further improve compliance, patients can be instructed totake their medication at a certain day of the week, where thecomposition is administered on a weekly schedule, or at a certain day ofthe month in case of a monthly schedule.

The length of the time intervals between each administration of acomposition of the present invention may vary. For example said timeintervals may be selected in function of the plasma levels. Theintervals may be shorter where the plasma levels of bedaquiline (and/orits active metabolite M2) are deemed too low, e.g. when these approachthe minimum plasma level specified hereinafter. The intervals may belonger where the plasma levels of bedaquiline (and/or its activemetabolite M2) are deemed too high. In one embodiment, the compositionsof the invention are administered at equal time intervals. Thecompositions may be administered without any interjacent additionaladministrations, or with other words, the compositions may beadministered at particular points in time separated from one another bya time period of varying or equal length, e.g. a time period of at leastone week, or any other time period specified herein, during which nofurther bedaquiline is administered. Having time intervals of the samelength has the advantage that the administration schedule is simple,e.g. administration takes place at the same day in the week, or the sameday in the month. Such administration schedule therefore involveslimited “pill burden” thereby contributing beneficially to the patient'scompliance to the prescribed dosing regimen.

The concentration (or “C”) of bedaquiline (and/or its active metaboliteM2) in the plasma of a subject treated therewith is generally expressedas mass per unit volume, typically nanograms per milliliter (ng/ml). Forconvenience, this concentration may be referred to herein as “plasmadrug concentration” or “plasma concentration”.

The dose (or amount) of bedaquiline administered, depends on the amountof bedaquiline in the pharmaceutical compositions of the invention, oron the amount of a given composition that is administered. Where higherplasma levels are desired, either or both of a composition of higherbedaquiline concentration, or more of a given composition, may beadministered. This applies vice versa if lower plasma levels aredesired. Also a combination of varying time intervals and varying dosingmay be selected to attain certain desired plasma levels.

The dose (or amount) of bedaquiline administered also depends on thefrequency of the administrations (i.e. the time interval between eachadministration). Usually, the dose will be higher where administrationsare less frequent. All these parameters can be used to direct the plasmalevels to desired values

The dosing regimen also depends on whether prevention or treatment ofthe pathogenic mycobacterial infection is envisaged. In case of therapy,the dose of bedaquiline administered or the frequency of dosing, orboth, are selected so that the plasma concentration of bedaquiline iskept above a minimum plasma level. The term “minimum plasma level” (orC_(min)) in this context refers to the plasma level of bedaquiline(and/or its active metabolite M2) that provides effective treatment ofthe pathogenic mycobacterial infection. In particular, the plasma levelof bedaquiline (and/or its active metabolite M2) is kept at a levelabove a minimum plasma level of about 10 ng/ml, or above about 15 ng/ml,or above about 20 ng/ml, or above about 40 ng/ml. The plasma level ofbedaquiline (and/or its active metabolite M2) may be kept above aminimum plasma level that is higher, for example above about 50 ng/ml,or above about 90 ng/ml, or above about 270 ng/ml, or above about 540ng/ml. In one embodiment, the plasma level of bedaquiline (and/or itsactive metabolite M2) is kept above a level of about 13.5 ng/ml, or iskept above a level of about 20 ng/ml. Or the plasma level of bedaquiline(and/or its active metabolite M2) may be kept within certain ranges, inparticular ranges starting from a minimum plasma level selected fromthose mentioned above and ending at a higher plasma levels selected fromthose mentioned above and selected from 500 ng/ml and 1000 ng/ml (e.g.from 10 to 15, 10 to 20, 10 to 40, etc., or from 15 to 20, or 15 to 40,or 15 to 90, etc., or 20 to 40, 20 to 90, or 20 to 270, etc., or 40 to90, 40 to 270, or 40-540, etc., each time from about the indicated valuein ng/ml to about the indicated value in ng/ml). In one embodiment saidrange is from about 10 to about 20, from about 20 to about 90, from 90to 270, from 270 to 540, from 540 to 1000, each time from about theindicated value in ng/ml to about the indicated value in ng/ml.

The plasma levels of bedaquiline (and/or its active metabolite M2)should be kept above the above-mentioned minimum plasma levels becauseat lower levels the bacteria may no longer be sufficiently suppressed sothat it can multiply with the additional risk of the emergence ofmutations.

In the instance of prevention, the term “minimum plasma level” (orC_(min)) refers to the lowest plasma level of bedaquiline (and/or itsactive metabolite M2) that provides effective treatment/prevention ofinfection.

In particular, in the instance of prevention, the plasma level ofbedaquiline (and/or its active metabolite M2) can be kept at a levelabove a minimum plasma level mentioned above in relation to therapy.However in prevention the plasma level of bedaquiline (and/or its activemetabolite M2) can be kept at a lower level, for example at a levelabove about 4 ng/ml, or about 5 ng/ml, or about 8 ng/ml. The plasmalevels of bedaquiline (and/or its active metabolite M2) shouldpreferably be kept above these minimum plasma levels because at lowerlevels the drug may no longer be effective thereby increasing the riskof transmission of infection. Plasma levels of bedaquiline (and/or itsactive metabolite M2) may be kept at somewhat higher levels to have asafety margin. Such higher levels start from about 50 ng/ml or more. Theplasma level of bedaquiline (and/or its active metabolite M2) can bekept at a level that is in the ranges mentioned above in relation totherapy, but where the lower limits include the plasma levels of about 4ng/ml, or about 5 ng/ml, or about 8 ng/ml.

An advantage of bedaquiline (and/or its active metabolite M2) is that itmay be used up to relatively high plasma levels without any significantside effects. The plasma concentrations of bedaquiline (and/or itsactive metabolite M2) may reach relatively high levels, but as with anydrug should not exceed a maximum plasma level (or C_(max)), which is theplasma level where bedaquiline (and/or its active metabolite M2) causessignificant side effects. Additionally, compound-release from the tissueshould also be taken into account, which is not counted for withinplasma levels. As used herein, the term “significant side effects” meansthat the side effects are present in a relevant patient population to anextend that the side effects affect the patients' normal functioning. Inan embodiment, the amount and the frequency of administrations ofbedaquiline (and/or its active metabolite M2) to be administered areselected such that the plasma concentrations are kept during a long termat a level comprised between a maximum plasma level (or C_(max) asspecified above) and a minimum plasma level (or C_(min) as specifiedabove).

In certain instances it may be desirable to keep the plasma levels ofbedaquiline (and/or its active metabolite M2) at relatively low levels,e.g. as close as possible to the minimum plasma levels specified herein.This will allow reducing the frequency of the administrations and/or thequantity of bedaquiline (and/or its active metabolite M2) administeredwith each administration. It will also allow avoiding undesirable sideeffects, which will contribute to the acceptance of the dosage forms inmost of the targeted population groups who are healthy people at risk ofbeing infected and therefore are less inclined to tolerate side effects.The plasma levels of bedaquiline (and/or its active metabolite M2) maybe kept at relatively low levels in the instance of prevention. Oneembodiment concerns uses or methods for prevention of infection, asspecified above or hereinafter, wherein the minimum plasma level ofbedaquiline (and/or its active metabolite M2) is as specified herein andthe maximum plasma level is about equal to the lowest plasma level thatcauses the active ingredient to act therapeutically, also as specifiedherein.

In other embodiments, the plasma level of bedaquiline (and/or its activemetabolite M2) is kept at a level below a lower maximum plasma level ofabout 10 ng/ml, more in particular about 15 ng/ml, further in particularabout 20 ng/ml, still more in particular about 40 ng/ml. In a particularembodiment, the plasma level of bedaquiline (and/or its activemetabolite M2) is kept below a level of about 13.5 ng/ml. In oneembodiment, the plasma level of bedaquiline (and/or its activemetabolite M2) is kept in an interval of the lower maximum blood levelspecified above, and the minimum plasma levels mentioned in relation toprevention. For example the plasma levels of bedaquiline (and/or itsactive metabolite M2) are kept below about 10 ng/ml and above a minimumlevel of about 4 ng/ml.

In other instances it may be desirable to keep the plasma levels ofbedaquiline (and/or its active metabolite M2) at relatively higherlevels, for example where there is a high risk of infection and morefrequent and/or higher doses are not an issue. In these instances theminimum plasma level may be equal to the lowest plasma level ofbedaquiline (and/or its active metabolite M2) that provides effectivetreatment of a pathogenic mycobacterial infection, such as the specificlevels mentioned herein.

In the instance of prevention, the dose to be administered should becalculated on a basis of about 0.2 mg/day to about 50 mg/day, or 0.5mg/day to about 50 mg/day, or of about 1 mg/day to about 10 mg/day, orabout 2 mg/day to about 5 mg/day, e.g. about 3 mg/day. This correspondsto a weekly dose of about 1.5 mg to about 350 mg, in particular of about3.5 mg to about 350 mg, in particular of about 7 mg to about 70 mg, orabout 14 mg to about 35 mg, e.g. about 35 mg, or to a monthly dose offrom 6 mg to about 3000 mg, in particular about 15 mg to about 1,500 mg,more in particular of about 30 mg to about 300 mg, or about 60 mg toabout 150 mg, e.g. about 150 mg. Doses for other dosing regimens canreadily be calculated by multiplying the daily dose with the number ofdays between each administration.

In the instance of therapy, the dose to be administered should besomewhat higher and should be calculated on a basis of about 1 mg/day toabout 150 mg/day, or of about 2 mg/day to about 100 mg/day, or of about5 mg/day to about 50 mg/day, or about 10 mg/day to about 25 mg/day, e.g.about 15 mg/day. The corresponding weekly or monthly doses can becalculated as set forth above. For applications in prevention, the dosesmay be lower although the same dosing as for therapeutic applicationsmay be used. In an embodiment, the dose/administration is given atmonthly intervals or three-monthly or six-monthly intervals, with thetotal treatment duration being three, six or 12 months. In the instanceswhere the dose/administration is monthly, three monthly or six-monthly,in an embodiment, the dose given (e.g. in human subjects) is calculatedon the basis of a 400 mg daily dose given for 2 weeks. Hence, the totalamount of bedaquiline given per dose may be about 5600 mg (e.g. in therange of 3000 and 8000 mg), but it may be up to one fifth of such anamount (e.g. in the range of 500 and 2000 mg, e.g. between about 1000and 1500 mg).

In another embodiment, in the case of prevention or in particulartherapy, the doses may also be expressed in mg/kg. For instance, in theexamples, certain doses may be administered based on weight (of e.g. themammal, and as shown in the examples here, in mouse) and hence dosesbetween 1 mg/kg and 1000 mg/kg may be employed (e.g. 40 mg/kg, 80 mg/kg,160 mg/kg, 320 mg/kg or 480 mg/kg may be employed) and such doses mayremain effective for a period of 4 weeks, 8 weeks or 12 weeks (forexample as shown in the examples). For instance, one dose may be takenevery 4 weeks (effectively seen as a 12 week treatment regimen, i.e.three doses in total) or one single dose may be taken, which effectivelyprovides sufficient treatment (e.g. as defined by reduction in CFUs, seeexamples) as may be evidenced by monitoring over a 12 week period.Hence, in an aspect, in order to treat the bacterial infection one dosemay be taken (e.g. between 1 mg/kg and 1000 mg/kg, for instance between2 mg/kg and 500 mg/kg) or one such dose may be taken every 4 weeks (e.g.two or three such doses may be taken). Such dose depends on thebacterial infection to be treated. For instance, in the treatment oflatent tuberculosis or leprosy, lower doses may be required (compared toe.g. multi-drug resistant tuberculosis) given that a lower amount ofbedaquiline is required to control the bacteria. An example of this isdescribed hereinafter (Example 3), wherein it is indicated that in miceone dose of 160 mg/kg may sufficiently reduce CFUs in the mouse model oflatent tuberculosis infection—it was also seen that two or three dosesof 160 mg/kg (the second and the third doses administered at 4 and 8weeks, respectively) were also effective in that model.

It has been found that, once administered, the plasma levels ofbedaquiline (and/or its active metabolite M2) are more or less stable,i.e. they fluctuate within limited margins. The plasma levels have beenfound to approach more or less a steady state mode or to approximatemore or less a zero order release rate during a prolonged period oftime. By “steady state” is meant the condition in which the amount ofdrug present in the plasma of a subject stays at more or less the samelevel over a prolonged period of time. The plasma levels of bedaquiline(and/or its active metabolite M2) generally do not show any drops belowthe minimum plasma level at which the drug is effective. The term “staysat more or less the same level” does not exclude that there can be smallfluctuations of the plasma concentrations within an acceptable range,e.g. fluctuations within a range of about ±30%, or about ±20%, or about±10%, or about ±10%.

In some instances there may be an initial plasma concentration peakafter administration, after which the plasma levels achieve a“steady-state”, as mentioned hereinafter.

The compositions of the invention show good local tolerance and ease ofadministration. Good local tolerance relates to minimal irritation andinflammation at the site of injection; ease of administration refers tothe size of needle and length of time required to administer a dose of aparticular drug formulation. In addition, the compositions of theinvention show good stability and have an acceptable shelf life.

The micro- or nanoparticles of the present invention have a surfacemodifier adsorbed on the surface thereof. The function of the surfacemodifier is to act as a wetting agent as well as a stabilizer of thecolloidial suspension.

In one embodiment, the micro- or nanoparticles in the compositions ofthe invention mainly comprise crystalline bedaquiline or a salt thereof;and a surface modifier, the combined amount of which may at leastcomprise about 50%, or at least about 80%, or at least about 90%, or atleast about 95%, or at least about 99% of the micro- or nano particles.As indicated herein, in an embodiment, bedaquiline is in its non-saltform (or in its “free form”) and in a further embodiment it is in acrystalline non-salt (or free) form. In this respect, as mentionedherein, bedaquiline may be prepared as such using the proceduresdescribed in international patent application WO 2004/011436 (or in WO2006/125769, which describes an optical resolution with a chiralreagent). Following such procedure, the bedaquiline is obtained byprecipitation from toluene/ethanol and it is indicated that the productcrystallises. Such form of bedaquiline may be used in the preparation ofthe compositions of the invention and, further, such form may be asingle crystalline polymorph with the following characterising features:

-   -   (i) a melting endotherm at 181.5° C. (endotherm onset) and DSC        curve showing melting of the product at about 182.5° C.        (immediately followed by decomposition; measured by differential        scanning calorimetry (DSC) by transfer of about 3 mg of compound        into a standard aluminum TA-Instrument sample pan, sample pan        closed with the appropriate coer and DSC curve recorded on a        TA-Instruments Q2000 MTDSC equipped with a RCS cooling unit        using the following parameters—initial temperature 25° C.;        heating range 10° C./min; final temperature 300° C., nitrogen        flow 50 ml/min);    -   (ii) infrared (IR) spectrum peaks at inter alia about 1600 cm⁻¹,        about 1450 cm⁻¹, about 1400 cm⁻¹, about 1340 cm⁻¹, and about        1250 cm⁻¹ (where a sample is analysed using a suitable microATR        accessory deploying 32 scans, 1 cm⁻¹ resolution, Thermo Nexus        670 FTIR spectrometer, a DTGS with KBr windows detector, Ge on        KBr beamsplitter and a micro ATR accessory (Harrick Split Pea        with Si crystal); and/or    -   (iii) X-ray powder diffraction (XRPD) with characteristic peaks        at about 11.25° 2-Theta, about 18° 2-Theta, about 18.5° 2-Theta,        about 19° 2-Theta, about 20.25° 2-Theta, about 21.25° 2-Theta,        about 22.25° 2-Theta, about 24.5° 2-Theta and about 27° 2-Theta,        showing diffraction peaks without the presence of a halo        indicating crystallinity of the product (where the analysis was        carried out on a PANalytical (Philips) X'PertPRO MPD        diffractometer, and the instrument is equipped with a Cu LFF        X-ray tube and the compound was spread on a zero background        sample holder; the Instrument Parameters were: generator        voltage—45 kV; generator amperage—40 mA;        geometry—Bragg-Brentano; stage—spinner stage; scan        mode—continuous; scan range 3 to 50° 2θ; step size 0.02°/step;        counting time 30 sec/step; spinner revolution time—1 sec;        radiation type CuKα).        Hence, in an embodiment, the bedaquiline employed in a process        to prepare compositions of the invention (i.e. before conversion        to micro/nano-particles) is a crystalline form (e.g. of the        specific form characterised above). In a further embodiment of        the invention, the bedaquiline employed in the compositions of        the invention (i.e. after conversion to micro/nano-particles,        for instance by milling) is also in a crystalline form (e.g. of        the specific form characterised above).

In a further aspect, the present invention is concerned with apharmaceutical composition for administration by intramuscular orsubcutaneous injection, comprising a therapeutically effective amount ofbedaquiline, or a pharmaceutically acceptable salt thereof, in the formof a suspension of particles consisting essentially of:

-   (1) bedaquiline, or a pharmaceutically acceptable salt thereof in    micro- or nanoparticle form, having a surface modifier adsorbed to    the surface thereof; and-   (2) a pharmaceutically acceptable aqueous carrier; wherein the    active ingredient is suspended.

Suitable surface modifiers can be selected from known organic andinorganic pharmaceutical excipients, including various polymers, lowmolecular weight oligomers, natural products and surfactants. Particularsurface modifiers include nonionic and anionic surfactants.Representative examples of surface modifiers include gelatin, casein,lecithin, salts of negatively charged phospholipids or the acid formthereof (such as phosphatidyl glycerol, phosphatidyl inosite,phosphatidyl serine, phosphatic acid, and their salts such as alkalimetal salts, e.g. their sodium salts, for example egg phosphatidylglycerol sodium, such as the product available under the tradenameLipoid™ EPG), gum acacia, stearic acid, benzalkonium chloride,polyoxyethylene alkyl ethers, e.g., macrogol ethers such as cetomacrogol1000, polyoxyethylene castor oil derivatives; polyoxyethylene stearates,colloidal silicon dioxide, sodium dodecylsulfate, carboxymethylcellulosesodium, bile salts such as sodium taurocholate, sodiumdesoxytaurocholate, sodium desoxycholate; methylcellulose,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropyl-methylcellulose, magnesium aluminate silicate, polyvinylalcohol (PVA), poloxamers, such as Pluronic™ F68, F108 and F127 whichare block copolymers of ethylene oxide and propylene oxide; tyloxapol;Vitamin E-TGPS (α-tocopheryl polyethylene glycol succinate, inparticular α-tocopheryl polyethylene glycol 1000 succinate);poloxamines, such as Tetronic™ 908 (T908) which is a tetrafunctionalblock copolymer derived from sequential addition of ethylene oxide andpropylene oxide to ethylenediamine; dextran; lecithin; dioctyl ester ofsodium sulfosuccinic acid such as the products sold under the tradenameAerosol OT™ (AOT); sodium lauryl sulfate (Duponol™ P); alkyl arylpolyether sulfonate available under the tradename Triton™ X-200;polyoxyethylene sorbitan fatty acid esters (Tweens™ 20, 40, 60 and 80);sorbitan esters of fatty acids (Span™ 20, 40, 60 and 80 or Arlacel™ 20,40, 60 and 80); polyethylene glycols (such as those sold under thetradename Carbowax™ 3550 and 934); sucrose stearate and sucrosedistearate mixtures such as the product available under the tradenameCrodesta™ F110 or Crodesta™ SL-40; hexyldecyl trimethyl ammoniumchloride (CTAC); polyvinylpyrrolidone (PVP). If desired, two or moresurface modifiers can be used in combination.

Particular surface modifiers are selected from poloxamers, α-tocopherylpolyethylene glycol succinates, polyoxyethylene sorbitan fatty acidesters, and salts of negatively charged phospholipids or the acid formthereof. More in particular the surface modifiers are selected fromPluronic™ F108, Vitamin E TGPS, Tween™ 80, and Lipoid™ EPG (and, in aparticular embodiment, it is Vitamin E TPGS). One or more of thesesurface modifiers may be used. Pluronic™ F108 corresponds to poloxamer338 and is the polyoxyethylene, polyoxypropylene block copolymer thatconforms generally to the formulaHO—[CH₂CH₂O]_(x)—[CH(CH₃)CH₂O]_(y)—[CH₂CH₂O]_(z)—H in which the averagevalues of x, y and z are respectively 128, 54 and 128. Other commercialnames of poloxamer 338 are Hodag Nonionic™ 1108-F and Synperonic™PE/F108. In one embodiment, the surface modifier comprises a combinationof a polyoxyethylene sorbitan fatty acid ester and a phosphatidylglycerol salt (in particular egg phosphatidyl glycerol sodium).

The optimal relative amount of bedaquiline in relation to the surfacemodifier depends on the surface modifier selected, the specific surfacearea of the bedaquiline suspension which is determined by the averageeffective particle size and the bedaquiline concentration, the criticalmicelle concentration of the surface modifier if it forms micelles, etc.The relative amount (w/w) of bedaquiline to the surface modifierpreferably is in the range of 1:2 to about 20:1, in particular in therange of 1:1 to about 10:1, e.g. about 4:1.

The particles of this invention can be prepared by means ofmicronization/particle size reduction/nanonization by mechanical meansand by controlled precipitation from a supersaturated solution, or byusing supercritical fluids such as in the GAS technique (“gasanti-solvent”), or any combination of such techniques. In one embodimenta method is used comprising the steps of dispersing bedaquiline in aliquid dispersion medium and applying mechanical means in the presenceof grinding media to reduce the particle size of bedaquiline to anaverage effective particle size of less than about 50 μm, in particularless than about 1,000 nm. The particles can be reduced in size in thepresence of a surface modifier.

A general procedure for preparing the particles of this inventioncomprises

-   (a) obtaining bedaquiline in micronized form;-   (b) adding the micronized bedaquiline to a liquid medium to form a    premix/predispersion; and-   (c) subjecting the premix to mechanical means in the presence of a    grinding medium to reduce the average effective particle size.

Bedaquiline in micronized form is prepared using techniques known in theart. It is preferred that the average effective particle size of thebedaquiline active agent in the predispersion be less than about 100 μmas determined by sieve analysis. Where the average effective particlesize of the micronized bedaquiline is greater than about 100 μm, it ispreferred that the particles of the bedaquiline compound be reduced insize to less than 100 μm (for example to a size or size range asdescribed herein).

The micronized bedaquiline can then be added to a liquid medium in whichit is essentially insoluble to form a predispersion. The concentrationof bedaquiline in the liquid medium (weight by weight percentage) canvary widely and depends on the selected surface modifier and otherfactors. Suitable concentrations of bedaquiline in compositions varybetween about 0.1% to about 60%, or between about 1% to about 60%, orbetween about 10% to about 50%, or between about 10% to about 30%, e.g.about 10%, 20% or 30% (each % in this paragraph relating to w/v).

The premix can be used directly by subjecting it to mechanical means toreduce the effective average effective particle size in the dispersionto less than 2,000 nm. It is preferred that the premix be used directlywhen a ball mill is used for attrition. Alternatively, bedaquiline and,optionally, the surface modifier, can be dispersed in the liquid mediumusing suitable agitation such as, for example, a roller mill, until ahomogeneous dispersion is achieved.

The mechanical means applied to reduce the effective average effectiveparticle size of bedaquiline conveniently can take the form of adispersion mill. Suitable dispersion mills include a ball mill, anattritor/attrition mill, a vibratory mill, a planetary mill, mediamills, such as a sand mill and a bead mill. A media mill is preferreddue to the relatively shorter milling time required to provide thedesired reduction in particle size. The beads preferably are ZrO₂ beads.For instance, for the nanoparticles, the ideal bead size is about 0.5 mmand, for the microparticles, the ideal bead size is about 2 mm.

The grinding media for the particle size reduction step can be selectedfrom rigid media preferably spherical or particulate in form having anaverage size less than 3 mm and, more preferably, less than 1 mm (as lowas 200 μm beads). Such media desirably can provide the particles of theinvention with shorter processing times and impart less wear to themilling equipment. Examples of grinding media are ZrO₂ such as 95% ZrO₂stabilized with magnesia or stabilized with yttrium, zirconium silicate,glass grinding media, polymeric beads, stainless steel, titania, aluminaand the like. Preferred grinding media have a density greater than 2.5g/cm³ and include 95% ZrO₂ stabilized with magnesia and polymeric beads.

The attrition time can vary widely and depends primarily upon theparticular mechanical means and processing conditions selected. Forrolling mills, processing times of up to two days or longer may berequired.

The particles should be reduced in size at a temperature that does notsignificantly degrade the bedaquiline compound. Processing temperaturesof less than 30 to 40° C. are ordinarily preferred. If desired, theprocessing equipment may be cooled with conventional cooling equipment.The method is conveniently carried out under conditions of ambienttemperature and at processing pressures, which are safe and effectivefor the milling process.

The pharmaceutical compositions according to the present inventioncontain an aqueous carrier that preferably is pharmaceuticallyacceptable. Said aqueous carrier comprises sterile water optionally inadmixture with other pharmaceutically acceptable ingredients. The lattercomprise any ingredients for use in injectable formulations. Suchingredients are optional. These ingredients may be selected from one ormore of a suspending agent, a buffer, a pH adjusting agent, apreservative, an isotonizing agent, and the like ingredients. In oneembodiment, said ingredients are selected from one or more of asuspending agent, a buffer, a pH adjusting agent, and optionally, apreservative and an isotonizing agent. Particular ingredients mayfunction as two or more of these agents simultaneously, e.g. behave likea preservative and a buffer, or behave like a buffer and an isotonizingagent.

Suitable optional buffering agents and pH adjusting agents should beused in amount sufficient to render the dispersion neutral to veryslightly basic (up to pH 8.5), preferably in the pH range of 7 to 7.5.Particular buffers are the salts of week acids. Buffering and pHadjusting agents that can be added may be selected from tartaric acid,maleic acid, glycine, sodium lactate/lactic acid, ascorbic acid, sodiumcitrates/citric acid, sodium acetate/acetic acid, sodiumbicarbonate/carbonic acid, sodium succinate/succinic acid, sodiumbenzoate/benzoic acid, sodium phosphates,tris(hydroxymethyl)aminomethane, sodium bicarbonate/sodium carbonate,ammonium hydroxide, benzene sulfonic acid, benzoate sodium/acid,diethanolamine, glucono delta lactone, hydrochloric acid, hydrogenbromide, lysine, methanesulfonic acid, monoethanolamine, sodiumhydroxide, tromethamine, gluconic, glyceric, gluratic, glutamic,ethylene diamine tetraacetic (EDTA), triethanolamine, including mixturesthereof. In an embodiment, the compositions of the invention do notcontain a buffering agent.

Suitable optional preservatives comprise antimicrobials andanti-oxidants which can be selected from the group consisting of benzoicacid, benzyl alcohol, butylated hydroxyanisole (BHA), butylatedhydroxytoluene (BHT), chlorbutol, a gallate, a hydroxybenzoate, EDTA,phenol, chlorocresol, metacresol, benzethonium chloride,myristyl-γ-piccolinium chloride, phenylmercuric acetate and thimerosal.Radical scavengers include BHA, BHT, Vitamin E and ascorbyl palmitate,and mixtures thereof. Oxygen scavengers include sodium ascorbate, sodiumsulfite, L-cysteine, acetylcysteine, methionine, thioglycerol, acetonesodium bisulfate, isoacorbic acid, hydroxypropyl cyclodextrin. Chelatingagents include sodium citrate, sodium EDTA and malic acid. In anembodiment of the invention, the compositions of the invention do notcontain a perseverative.

An isotonizing agent or isotonifier may be present to ensure isotonicityof the pharmaceutical compositions of the present invention, andincludes sugars such as glucose, dextrose, sucrose, fructose, trehalose,lactose; polyhydric sugar alcohols, preferably trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Alternatively, sodium chloride, sodium sulfate, or otherappropriate inorganic salts may be used to render the solutionsisotonic. These isotonifiers can be used alone or in combination. Thesuspensions conveniently comprise from 0 to 10% (w/v), in particular 0to 6% of isotonizing agent. Of interest are nonionic isotonifiers, e.g.glucose, as electrolytes may affect colloidal stability. In anembodiment of the invention, the compositions of the invention contain aisotonizing agent or isotonifier, which, in a further embodiment is anonionic isotonifier, such as a suitable sugar such as mannitol.

A desirable feature for a pharmaceutical composition of the inventionrelates to the ease of administration. The viscosity of thepharmaceutical compositions of the invention should be sufficiently lowto allow administration by injection. In particular they should bedesigned so that they can be taken up easily in a syringe (e.g. from avial), injected through a fine needle (e.g. a 20 G 1½, 21 G 1½, 22 G 2or 22 G 1¼ needle) in not too long a time span. In one embodiment theviscosity of the compositions of the invention is below about 75 mPa·s,or below 60 mPa·s. Aqueous suspensions of such viscosity or lowerusually meet the above-mentioned criteria.

Ideally, the aqueous suspensions according to the present invention willcomprise as much bedaquiline (or pharmaceutically acceptable saltthereof) as can be tolerated so as to keep the injected volume to aminimum, in particular from 3 to 70% (w/v), or from 3 to 60% (w/v), orfrom 3 to 40% (w/v), or from 10 to 40% (w/v), of bedaquiline (orpharmaceutically acceptable salt thereof). In one embodiment the aqueoussuspensions of the invention contain about 50%-70% (w/v) of bedaquiline(or pharmaceutically acceptable salt thereof), or about 40%-60% (w/v) ofbedaquiline (or pharmaceutically acceptable salt thereof), or about30%-50% (w/v) of bedaquiline (or pharmaceutically acceptable saltthereof).

In one embodiment, the aqueous suspensions may comprise by weight, basedon the total volume of the composition:

-   (a) from 10% to 70% (w/v), or from 20% to 60% (w/v), or from 20% to    50% (w/v), or from 20% to 40% (w/v) of bedaquiline (or    pharmaceutically acceptable salt thereof);-   (b) from 0.5% to 20%, or from 2% to 15% or 20% (w/v), or from 5% to    15% (w/v) of a wetting agent;-   (c) from 0% to 10%, or from 0% to 5%, or from 0% to 2%, or from 0%    to 1% of one or more buffering agents;-   (d) from 0% to 20%, or from 2% to 15% or 20% (w/v), or from 5% to    15% (w/v) of a isotonizing agent-   (e) from 0% to 2% (w/v) preservatives; and-   (f) water for injection q.s. ad 100%.

In one embodiment, the aqueous suspensions may comprise by weight, basedon the total volume of the composition:

-   (a) from 3% to 50% (w/v), or from 10% to 40% (w/v), or from 10% to    30% (w/v), of bedaquiline (or pharmaceutically acceptable salt    thereof);-   (b) from 0.5% to 10%, or from 0.5% to 2% (w/v) of a wetting agent;-   (c) from 0% to 10%, or from 0% to 5%, or from 0% to 2%, or from 0%    to 1% of one or more buffering agents;-   (d) from 0% to 10%, or from 0% to 6% (w/v) of a isotonizing agent-   (e) from 0% to 2% (w/v) preservatives; and-   (f) water for injection q.s. ad 100%.

To the suspensions may optionally be added an amount of acid or base tobring the pH to a value of about pH 7. Suitable acids or bases are anyof those that are physiologically acceptable, e.g. HCl, HBr, sulfuricacid, alkali metal hydroxides such as NaOH. In an embodiment, such acidor base need not be added to the compositions of the invention.

The administration of bedaquiline (or pharmaceutically acceptable saltthereof) as in the present invention may suffice to treat a pathogenicmycobacterial infection although in a number of cases it may berecommendable to co-administer other anti-TB drugs.

In certain instances, the treatment of a pathogenic mycobacterialinfection may be limited to only the administration of a composition ofbedaquiline (and/or its metabolite thereof) in accordance with thisinvention, i.e. as monotherapy without co-administration of furtheranti-TB drugs. This option may be recommended, for example, for certainmycobacterial infections where a low concentration of the activeingredient may treat the bacteria (e.g. for latent/dormant TB or forMycobacterium leprae).

In a further aspect the present invention relates to the use of apharmaceutical composition comprising an effective amount of bedaquilineor a pharmaceutically acceptable salt thereof, in accordance with thepresent invention, for the manufacture of a medicament for maintenancetherapy of a subject being infected with a pathogenic mycobacterialinfection, wherein the composition is administered or is to beadministered intermittently at a time interval that is in the range ofone week to one year, or one week to two years.

Thus in a further aspect, the present invention provides a method forthe long term treatment of a patient being infected with a pathogenicmycobacterial infection, said method comprising

-   (i) the treatment of said patient with a combination of anti-TB    drugs; followed by-   (ii) the intermittent administration of a pharmaceutical composition    comprising an effective amount of bedaquiline or a pharmaceutically    acceptable salt thereof, in accordance with the present invention,    wherein the composition is administered at a time interval of at    least one week.

Where, the treatment is directed towards Mycobacterium leprae, thenagain the treatment regime might be given as monotherapy or incombination with existing drugs useful for the treatment ofMycobacterium leprae (e.g. rifapentin). The composition of the inventionmight be administered by injection once, or up to three times, e.g. asmonthly intervals. Advantages are associated with compliance, noresistance by avoiding dapsone, no stigma by avoiding clofazimine.

The present invention also concerns a pharmaceutical composition asdescribed hereinbefore for use as a medicament in the treatment orprophylaxis of a pathogenic mycobacterial infection.

In addition, the present invention concerns the use of a pharmaceuticalcomposition as described herein for the preparation of a medicament forthe prophylaxis or treatment of a pathogenic mycobacterial infection.

The present invention further concerns a method of treating a subjectinfected with a pathogenic mycobacterial infection, said methodcomprising the administration of a therapeutically effective amount of apharmaceutical composition as described herein.

As used herein, the word “substantially” does not exclude “completely”e.g. a composition which is “substantially free” from Y may becompletely free from Y. Where necessary, the word “substantially” may beomitted from the definition of the invention. The term “about” inconnection with a numerical value is meant to have its usual meaning inthe context of the numerical value. Where necessary the word “about” maybe replaced by the numerical value ±10%, or ±5%, or ±2%, or ±1%. Alldocuments cited herein are incorporated by reference in their entirety.

The following examples are intended to illustrate the present inventionand should not be construed as limiting the invention thereto.

Example 1: Preparation of Micro- and Nano-Suspensions

The active ingredient bedaquiline may be used as such or may beconverted into a pharmaceutically acceptable salt thereof, such as afumarate salt (for example the form used in the marketed productSirturo®). Where referred to herein, bedaquiline is used in its non-saltform unless otherwise specified.

The prototype of the bedaquiline formulation is as follows:

Preparation of 200 and 100 mg/mL nano- and micro-suspensions.

Materials Used:

Zirconium beads 0.5 mm (to aid process)Sterile water for injection (Viaflo)Bedaquiline (not milled/ground)Tocopheryl PEG 1000 succinate—an excipientZirconium beads 2 mm (to aid process)Mannitol (parenteral)—an excipient

Glass bottles and ZrO₂ beads (either 0.5 mm or 2 mm, depending on thedesired nano- or micro-suspensions), used as the milling media, weresterilized in an autoclave. The drug substance (quantity depending onthe formulation to be prepared; see e.g. formulation/suspension below)was put into the glass bottle as well as a solution of Tocopheryl PEG1000 succinate in water (quantity depending on the concentrationrequired/desired; see e.g. formulation/suspension below) for injection.ZrO₂-beads with an average particle size of 500 μm or 2 mm (depending onwhether a micro- or nano-suspension is required/desired) were added. Thebottle was placed on a roller mill. The suspension wasmicronized/nanonized at 100 rpm for a period of time up to 72 hours. Forinstance, micronizing may be performed at 100 rpm for a period of 3hours (or up to 3 hours) and nanonizing may be performed at 100 rpm fora period of up to 46 hours (e.g. about 40 hours). At the end of themilling process the concentrated micro- or nano-suspension was removedwith a syringe and filled into vials. The resulting formulations (basedon the nano-suspension and micro-suspension) are described in thefollowing tables. Determination of the concentration was done byHPLC/UV. If needed, a dilution was made to a final concentration of 200mg/ml of active ingredient bedaquiline. The resulting suspension wasshielded from light. Other concentrations were also made and tested,including 300 mg/ml and 100 mg/ml nano- and micro-formulations.

Such formulations were (and will be) dosed intramuscular andsubcutaneous in animals for PK study to investigate a possiblelong-acting effect (e.g. in treatment of leprosy). Physical stability ofthe suspensions will be followed up by measuring particle size afterdifferent storage conditions.

Certain embodiments of the formulation(s) have the following features:

-   -   Micro-suspension by using 2 mm Zr beads    -   Milling at 200 mg/mL (otherwise the concentration may be too        high, e.g. with 300 mg/ml)    -   Longer milling, resulting in nano-suspension    -   A suitable surface modifier, for instance selected based on        physical stability, e.g. in one embodiment it is TPGS, and, in        another embodiment it is Tween

Examples of Bedaquiline Micro- and Nano-Suspensions

200 mg/ml Nano- and Micro-Suspension Referred to Herein as Example 1A(Nano) and Example 1B (Micro)

mg/ml Bedaquiline 200 TPGS  50 Mannitol  50 Sterile water for injectionq.s.100 mg/ml Nano- and Micro-Suspension Referred to Herein as Example 1C(Nano) and Example 1D (Micro)

mg/ml Bedaquiline 100 TPGS  25 Mannitol  50 Sterile water for injectionq.s.

Particle Size Distribution (PSD) of the Above Formulations

Where applicable, ND=not determined

PSD for 200 mg/ml micro-suspension (Example 1B)

Storage Storage D10 D50 D90 temperature (° C.) time (μm) (μm) (μm) Afterpreparation 1.316 3.283 9.623  5 3 days 1.256 2.539 5.991 14 days 1.1422.582 7.386 1 month 1.157 2.423 5.850 3 months 1.065 2.225 5.141 25 3days 1.150 2.348 5.447 14 days 1.073 2.308 5.824 1 month 1.098 2.3225.665 3 months 1.178 2.452 5.826 40 3 days 1.110 2.227 4.913 14 days1.054 2.211 5.115 1 month 1.182 2.254 4.626 3 months 0.998 1.89  3.734

PSD for 200 mg/ml nano-suspension (Example 1A)

Storage Storage D10 D50 D90 temperature (° C.) time (μm) (μm) (μm) Afterpreparation 0.074 0.175 1.693  5 3 days 0.076 0.185 1.920 14 days 0.0810.219 8.995 1 month 0.075 0.176 1.281 3 months 0.076 0.183 1.884 25 3days 0.111 41.364 226.147 14 days ND ND ND 1 month ND ND ND 3 months NDND ND 40 3 days 0.097 0.483 168.316 14 days 0.1  0.642 240.375 1 month0.089 0.294 63.986 3 months 0.088 0.274 4.279

PSD for 100 mg/ml micro-suspension (Example 1D)

Storage Storage D10 D50 D90 temperature (° C.) time (μm) (μm) (μm) Afterpreparation 1.267 2.557 6.236  5 3 days 1.157 2.376 5.506 14 days 0.1250.320 0.993 1 month 1.1  2.337 5.625 3 months 1.048 2.236 5.269 25 3days 0.697 1.906 4.324 14 days 0.151 1.770 4.351 1 month 0.171 1.7974.253 3 months 1.104 2.266 5.142 40 3 days 0.547 1.657 3.502 14 days0.203 1.709 3.881 1 month 1.016 1.996 4.199 3 months 1.025 1.936 3.867

PSD for 100 mg/ml nano-suspension (Example 1C)

Storage Storage D10 D50 D90 temperature (° C.) time (μm) (μm) (μm) Afterpreparation 0.072 0.159 0.576  5 3 days 0.074 0.173 0.765 14 days 0.0800.213 7.889 1 month 0.075 0.177 0.780 3 months 0.074 0.172 0.919 25 3days 0.076 0.181 0.872 14 days 0.080 0.202 1.351 1 month 0.080 0.2031.673 3 months 0.083 0.222 1.691 40 3 days 0.077 0.187 1.017 14 days0.082 0.226 2.893 1 month 0.084 0.235 2.356 3 months 0.084 0.239 2.472

Example 2: Pharmacokinetic Studies Study A—Pharmacokinetic Profile inMice

A single dose of bedaquiline was administered to the mouse orally andthe plasma kinetics of bedaquiline itself (also referred to as “TMC207”)and its main metabolite, N-monodesmethyl (also referred to as “M2”),were measured over a period of 168 hours. M2 seems to be an activemetabolite and its formation, upon administration of bedaquiline(TMC207), is seen in at least the following species: mouse, rat/dog andhuman (its formation being the most in mouse and least in humans).

The results are as described in FIG. 1: “Plasma kinetics of TMC207 andM2 in mouse, after a single dose of 30 mg/kg”

It could be seen that:

-   -   TMC207 and M2 plasma kinetics are slow; the formation of M2 is        also slow    -   M2 plasma exposure (AUC) is greater than the TMC207 exposure    -   M2 lung concentrations is a lot greater than TMC207 lung        concentrations    -   After a period of 168 hours, the concentration of TMC207 in the        plasma is about 0.01 μg/ml and of M2 is about 0.1 μg/ml

As described in Example 1, the 200 mg/ml and 100 mg/ml micro- andnano-suspensions (Examples 1A, 1B, 1C and 1D) were tested on mice, wherethe mice either received:

-   -   a dose of 80 mg/kg (in which case the 100 mg/ml suspensions were        used, i.e. Example 1C and 1D) or 160 mg/kg (in which case the        200 mg/ml suspensions were used, i.e. Example 1A and 1B)    -   were dosed intramuscularly (IM) or subcutaneously (SM)

Each of the formulations 1A, 1B, 1C and 1D were tested in a suspensionAPI assay before administering into the mice, and it was determined thatthe API was in the range of 75-142% (an unusually broad range). However,in the mice, the plasma levels of bedaquiline and its metabolite couldstill be measured and assessed after administering such formulations.

Phase 1 of the Results—Up to 672 Hours

FIG. 2 “Plasma kinetics of TMC207 in mouse when administered IM or SCwith 200 mg/ml formulations (specifically formulations of Examples 1Aand 1B, i.e. the nano- and micro-suspension, respectively) at a dose of160 mg/kg” (TMC207 is referred to the in the Figure as “UD”)

FIG. 3 “Plasma kinetics of M2 in mouse when administered IM or SC with200 mg/ml formulations (specifically formulations of Examples 1A and 1B,i.e. the nano- and micro-suspension, respectively) at a dose of 160mg/kg” (M2 is referred to in the Figure as “met”)

Generally, it can be seen that:

-   -   for the TMC207 concentrations, the C_(max) ranges from between        about 3000 ng/ml (the highest for the micro-suspension dosed IM)        to about 100 ng/ml (the lowest being for the micro-suspension        dosed SC)    -   at 672 hours, there was still a measureable concentration of        TMC207 ranging from about 200 ng/ml (the highest for the        micro-suspension dosed IM) to about 50 ng/ml (for lowest for the        micro-suspension dosed SC)    -   for the M2 concentrations, the C_(max) ranges from between about        3000 ng/ml (the highest for the micro-suspension dosed IM) to        about 300 ng/ml (the lowest for the micro-suspension dosed SC)    -   at 672 hours, there was still a measureable concentration of M2        ranging from about 1000 ng/ml (the highest for the        micro-suspension dosed IM) to about 200 ng/ml (the lowest for        the micro-suspension dosed SC)

FIG. 4 “Plasma kinetics of TMC207 in mouse when administered IM or SCwith 100 mg/ml formulations (specifically formulations of Examples 1Cand 1D, i.e. the nano- and micro-suspension, respectively) at a dose of80 mg/kg” (TMC207 is referred to the in the Figure as “UD”)

FIG. 5 “Plasma kinetics of M2 in mouse when administered IM or SC with100 mg/ml formulations (specifically formulations of Examples 1C and 1D,i.e. the nano- and micro-suspension, respectively) at a dose of 80mg/kg” (M2 is referred to in the Figure as “met”)

Generally, it can be seen that:

-   -   for the TMC207 concentrations, the C_(max) ranges from between        about 2000 ng/ml (the highest for the nano-suspension dosed IM)        to about 400 ng/ml (the lowest being for the nano- and        micro-suspension dosed SC)    -   at 672 hours, there was still a measureable concentration of        TMC207 ranging from about 100 ng/ml (the highest for the        micro-suspension dosed IM) to about 30 ng/ml (for lowest for the        micro-suspension dosed SC)    -   for the M2 concentrations, the C_(max) ranges from between about        2000 ng/ml (the highest for the nano-suspension dosed IM) to        about 300 ng/ml (the lowest for the micro-suspension dosed SC)    -   at 672 hours, there was still a measureable concentration of M2        ranging from about 500 ng/ml (the highest for the        micro-suspension dosed IM) to about 100 ng/ml (the lowest for        the micro-suspension dosed SC)

Phase 2 of the Results—Up to 2184 Hours

The mice of these studies were further monitored up to 2184 hours,giving the following results:

-   -   for Formulation 1A, i.e. the nano-suspension of 200 mg/ml        concentration, and dosed SC at 160 mg/kg (StDev=standard        deviation) and IM at 160 mg/kg

Plasma concentration of bedaquiline (BDQ) or its metabolite (M2) SC at160 mg/kg IM at 160 mg/kg Time (h) BDQ StDev M2 StDev BDQ StDev M2 StDev1 493 305 59.3 49.6 1517 710 171 71 4 676 384 284 188 1588 662 708 332 7728 269 484 312 1408 519 1063 456 24 726 53 956 289 1022 299 2071 828168 239 28 1240 475 219 63 1399 557 336 138 66 759 282 99.0 33.2 597 301504 122 53 503 178 66.1 26.1 418 209 672 109 22 383 136 79.0 34.8 405211 840 100.8 42.5 196.0 76.5 69.6 58.5 119.2 60.3 1176 70.3 31.0 117.850.0 34.7 13.8 65.7 30.9 1512 58.5 20.1 91.2 42.5 23.4 8.4 40.3 18.11848 40.6 16.6 86.3 41.7 17.4 7.3 29.7 15.8 2184 35.2 21.4 65.1 36.614.5 6.7 27.3 13.2 T max (h) 4-24 168 1-4 24 Cmax 862 202 1240 475 1723764 2071 828 (ng/mL) t1/2 (h) 910 442 1024 573 964 572 794 403 AUClast247783 54315 702286 173934 207109 69394 696089 302612 (ng*h/mL) AUCinf301991 107533 815741 282889 231176 89366 728091 318248 (ng*h/mL)

-   -   for Formulation 1C, i.e. the nano-suspension of 100 mg/ml        concentration, and dosed SC at 80 mg/kg and IM at 80 mg/kg

Plasma concentration of bedaquiline (BDQ) or its metabolite (M2) SC at80 mg/kg IM at 80 mg/kg Time (h) BDQ StDev M2 StDev BDQ StDev M2 StDev 1261 66 22.0 3.5 1515 568 177 51 4 538 288 222 88 1572 470 684 239 7 480281 342 108 1458 314 1049 256 24 205 109 545 185 1114 299 2186 834 16865.6 30.9 298 130 186 46 1393 744 336 42.5 21.5 192 122 89.0 23.1 609342 504 46.3 38.3 178 165 54.9 15.0 400 198 672 41.9 36.9 145 136 57.227.3 285 121 840 33.9 20.3 70.5 59.2 52.6 40.5 107 54 1176 25.4 15.947.2 37.5 28.2 17.8 50.4 29.9 1512 22.2 12.5 43.8 31.7 17.0 11.0 33.821.8 1848 14.1 7.3 28.8 18.8 12.6 9.1 23.2 17.4 2184 12.9 6.8 24.1 14.37.28 18.3 14.2 T max (h) 4-7 24 1-7 24 Cmax 557 265 545 185 1806 4732186 834 (ng/mL) t1/2 (h) 1051 390 796 147 581 159 650 122 AUClast 8412143933 238239 136814 186882 61016 669899 325833 (ng*h/mL) AUCinf 10298547867 264292 149139 196358 68907 688601 344341 (ng*h/mL)

-   -   for Formulation 1B, i.e. the micro-suspension of 200 mg/ml        concentration, and dosed SC at 160 mg/kg (StDev=standard        deviation) and IM at 160 mg/kg

Plasma concentration of bedaquiline (BDQ) or its metabolite (M2) SC at160 mg/kg IM at 160 mg/kg Time (h) BDQ StDev M2 StDev BDQ StDev M2 StDev1 71.1 15.1 6.53 2.02 1737 1752 206 247 4 101 7 37.5 7.1 2258 1229 908734 7 102 12 55.3 17.7 1764 1103 1474 795 24 130 19 186 36 1306 407 29261143 168 78.7 5.3 276 61 391 137 2643 1087 336 53.8 3.5 226 45 293 1311693 798 504 51.1 8.3 196 37 222 101 1526 773 672 67.4 12.8 266 52 231115 1202 680 840 65.7 28.9 114.3 36.6 163.5 74.1 387.8 198.0 1176 55.136.2 104.6 51.3 121.9 48.7 255.0 137.4 1512 38.2 13.6 95.6 34.4 94.060.7 165.2 91.2 1848 36.9 11.0 66.7 22.8 65.6 23.3 146.4 80.8 2184 30.710.4 60.9 21.9 51.5 26.1 112 62 T max (h) 24 168-672 1-4 24-168 Cmax 13019 309 30 2364 1447 3002 1139 (ng/mL) t1/2 (h) 1545 253 1294 383 719 70848 273 AUClast 117569 30034 300334 63219 447361 174979 1689422 755169(ng*h/mL) AUCinf 188153 60663 412456 107222 500850 201928 1823672 836740(ng*h/mL)

-   -   for Formulation 1D, i.e. the micro-suspension of 100 mg/ml        concentration, and dosed SC at 80 mg/kg (StDev=standard        deviation) and IM at 80 mg/kg

Plasma concentration of bedaquiline (BDQ) or its metabolite (M2) SC at80 mg/kg IM at 80 mg/kg Time (h) BDQ StDev M2 StDev BDQ StDev M2 StDev 1133 114 6.70 463 186 29.7 12.1 4 415 533 130 175 873 221 264 41 7 350412 232 310 850 200 459 60 24 162 47 360 364 709 228 1101 341 168 53.218.3 226 112 209 44 1050 405 336 28.7 6.8 107 35 112 12 547 150 504 28.20.8 109 27 71.8 17.5 398 121 672 28.6 5.8 105 33 87.0 17.0 444 127 84025.9 6.8 56.3 22.9 70.9 19.6 130 36 1176 25.3 4.5 42.9 15.8 41.5 8.491.2 27.4 1512 20.2 6.9 42.1 16.4 31.1 9.5 68.8 26.8 1848 19.6 8.0 31.514.7 24.0 7.0 43.7 15.2 2184 15.2 5.4 32.7 16.1 26.3 14.2 42.2 16.7 Tmax (h) 4-24 24-168 4-7 24-168 Cmax 433 517 383 348 925 192 1139 366(ng/mL) t1/2 (h) 1423 535 1082 437 916 337 734 322 AUClast 69275 7996175236 48413 192325 36480 586546 165428 (ng*h/mL) AUCinf 103115 29066226669 50867 225966 51489 632846 193499 (ng*h/mL)

Study B—Pharmacokinetic Profile in Rats and Beagle Dogs

Formulations of concentrations 200 mg/mL were used in this study, boththe nano-suspension (Formulation 1A) and the micro-suspension(Formulation 1B), as depicted above in Example 1 (i.e. using, inaddition to the 200 mg/ml concentration of micro- and nano-particles (ofthe active bedaquiline), TPGS (4:1 bedaquiline:TPGS) and 50 mg/mlMannitol in WFI (water for injection)).

These studies demonstrate that formulations described in Example 1(specifically the nano- and micro-formulations 1A and 1B) results instable plasma levels over a prolonged period of time in male rats andmale beagle dogs, when administered subcutaneously (SC) andintramuscularly (IM).

Male Rats

The first experiment was performed on male rats, where each relevant 200mg/ml nano-suspension and micro-suspension referred to above wereadministered subcutaneously (SC) and intramuscularly (IM) at aconcentration of 40 mg/kg (0.2 mL/kg). An interim analysis was performedat 3 months and the results were followed-up at 6 months. Twelve ratswere used in the study. Six rats were dosed intramuscularly (IM), threeof those rats with the 200 mg/ml nano-suspension (see Example 1,Formulation 1A above) and the other three with the 200 mg/mlmicro-suspension (see Example 1, Formulation 1B above). Six rats weredosed subcutaneously (SC), three of those rats with the 200 mg/mlnano-suspension (see Formulation 1A above) and the other three with the200 mg/ml micro-suspension (see Formulation 1B above).

Phase 1 of the Results—Up to 2200 Hours

FIG. 6 “Plasma kinetics of TMC207 in male rats when administered IM orSC with 200 mg/ml micro-formulation (see Example 1, Formulation 1B i.e.the micro-suspension) at a dose of 40 mg/kg” and “Plasma kinetics ofTMC207 in male rats when administered IM or SC with 200 mg/mlnano-formulation (see Example 1, Formulation 1A, i.e. thenano-suspension) at a dose of 40 mg/kg”

The following parameters were calculated for TMC207 (see FIG. 6):

Microsuspension Microsuspension Nanosuspension Nanosuspension (Form IB)SC (Form IB) IM (Form 1A) SC (Form 1A) IM n 3 3 3 3 C_(max) 68.1 ± 17.6 215 ± 66.7  337 ± 57.0  505 ± 96.6 (ng/ml) T_(max) ^(a) (h)  24  18  24 16 (24.00-24.00)  (7.00-24.00) (24.00-24.00)  (1.00-24.00) T_(last)^(a) = 2184 2184 2184 2184 around 3 (2184-2184) (2184-2184) (2184-2184)(2184-2184) mths (h) AUC_(0-2184h) 34700 ± 1770  91500 ± 13200 75400 ±5070  77900 ± 8930  (3 mths) (ng · h/ml)where applicable mean values are given (with min→max in parentheses)

Generally, it can be seen that:

-   -   after microsuspension administration, higher (2.6 fold) AUC        after IM versus SC. After nanosuspension administration, similar        AUC after SC or IM    -   in terms of bioavailability (comparison with IV 5 mg/kg), for        the lowest (microsuspension SC)=56%, for the 3 other >100%    -   M2, which is not specified on the graphs in FIG. 6, has the same        profiles as TMC207 except that t_(max) is later, AUC of M2 is        1.5 to 2 fold lower than TMC207; in general this ratio is        comparable to PO route

A comparison was also performed with oral (PO) administration in rats,which can also be considered a 13 week toxicity study, where thefollowing result was observed:

-   -   The exposures (C_(max) and AUC) at 3 months after single IM or        SC for both formulations are much lower than the total exposure        after PO administration at the top dose of 13 week study: IM/SC        34500-91500 ng·h/mL versus PO a total exposure=2 385 383 ng·h/mL        during the same period of time (3 months)    -   see above regarding M2

Male Beagle Dogs

The second experiment was performed on male beagle dogs, where eachrelevant 200 mg/ml nano-suspension and micro-suspension referred toabove were administered subcutaneously (SC) and intramuscularly (IM) ata concentration of 40 mg/kg (0.2 mL/kg). An interim analysis wasperformed at 3 months and the results were followed-up at 6 months.Twelve (12) healthy male beagle dogs with body weights ranging from 8 to16 kg at the start of the study, were used. Each dog was identified byan ear tattoo number. Six dogs were dosed intramuscularly (IM) in theleft and right m. biceps femoris, three of those dogs with the 200 mg/mlnano-suspension (see Example 1, Formulation 1A above) and the otherthree with the 200 mg/ml micro-suspension (see Example 1, Formulation1B). Six dogs were dosed subcutaneously (SC) in the left and rightthoracal region, three of those dogs with the 200 mg/ml nano-suspension(see Formulation 1A above) and the other three with the 200 mg/mlmicro-suspension (see Formulation 1B above).

Blood samples of 3 ml were taken from the left jugular vein from alldogs on day 0 at 0 h (predose), 20 min, 1 h, 3 h, 8 h and 24 h post-doseand further on days 2, 3, 6, 8, 10, 13, 16, 20, 23, 27, 29, 36, 43, 50,57, 64, 71, 78, 85 and 92 at approximately 8 AM. Blood samples wereplaced on EDTA, EDTA Vacuette Greiner, Cat. No. 454086, GreinerLabortechnik N.V.). Within 2 h of blood sampling, samples werecentrifuged at room temperature at about 1900×g for 10 minutes to allowplasma separation. Plasma was immediately transferred into a second tubeand stored in the freezer within 2 hours after the start ofcentrifugation. Plasma samples were analysed individually for TMC207,and for its metabolite M2, by means of a validated LC-MS/MS-method.

FIG. 7 “Plasma kinetics of TMC207 in male beagle dogs when administeredIM or SC with 200 mg/ml micro-formulation (see Example 1, Formulation1B) at a dose of 40 mg/kg” and “Plasma kinetics of TMC207 in male beagledogs when administered IM or SC with 200 mg/ml nano-formulation (seeExample 1, Formulation 1A) at a dose of 40 mg/kg”

The following parameters were calculated for TMC207 (see FIG. 7):

Microsuspension Microsuspension Nanosuspension Nanosuspension (Form 1B)SC (Form 1B) IM (Form 1A) SC (Form 1B) IM n 3 3 3 3 C_(max) 219 ± 237822 ± 211 692 ± 217 4150 ± 1290 (ng/ml) T_(max) ^(a) (h)  620 3.0  1682.0 (168.00-840.00) (1.00-7.00) (168.00-168.00) (1.00-4.00) T_(last)^(a) = 2184 2184 2184 2184 around 3 (2184-2184) (2184-2184) (2184-2184)(2184-2184) mths (h) AUC_(last) 268000 ± 250000 519000 ± 64300  483000 ±65300  549000 ± 26200  (ng · h/ml)where applicable mean values are given (with min→max in parentheses)

Generally, it can be seen that:

-   -   after microsuspension administration, higher (2 fold) AUC after        IM versus SC    -   after nanosuspension administration, similar AUC after SC or IM    -   in terms of C_(max) higher after IM versus SC for both        formulation    -   in terms of bioavailability (comparison with IV 1 mg/kg)>100%    -   M2 has the same profiles as TMC207 except that t_(max) is later,        AUC is 3 to 4 fold lower than TMC207; in general this ratio is        comparable to PO route

A comparison was also performed with oral (PO) administration in rats,which can be considered as a 13 week toxicity study, where the followingresult was observed:

-   -   The highest C_(max) after IM nanosuspension similar to the        C_(max) after PO at 18 mg/kg; in terms of exposure much higher        total exposure after PO versus after IM/SC: IM/SC 268000-549000        ng·h/mL versus PO a total exposure=13 988 520 ng·h/mL for the        same period    -   See above for M2

Based on the 3-month interim results, we have the following conclusions:

After IM/SC nanosuspension/microsuspesion:

-   -   In rats, AUC: IM micro>SCnano˜=IM nano (more rapid decline)>SC        micro    -   In dogs, AUC: IM micro>SCnano˜=IM nano>SC micro (similar decline        for the 4 profiles)

At 40 mg/kg after IM/SC nanosuspension/microsuspension, C_(max) and AUCof TMC207/M2 are covered by oral tox studies in both species except forthe C_(max) of TMC207 in dogs after IM nanosuspension which is similarbetween PO and IM

Phase 2 of the Results—Up to 4400 Hours

In all cases the plasma concentration of BDQ or M2 is calculated as themean of the three animals (rats or dogs) in the relevant study.

Study in rats: for Formulation 1B, i.e. the micro-suspension of 200mg/ml concentration, and dosed SC at 40 mg/kg (StDev=standard deviation)and IM at 40 mg/kg

Plasma concentration of bedaquiline (BDQ) or its metabolite (M2) SC at40 mg/kg IM at 40 mg/kg Time (h) BDQ StDev M2 StDev BDQ StDev M2 StDev 122.1 5.36 0.589 NC 139 33.2 5.11 2.24 4 36.9 7.42 6.62 1.69 172 47.218.8 5.98 7 40.7 6.27 8.59 1.46 185 24.2 28.7 7.57 24 68.1 17.6 24.01.14 212 70.6 77.3 23.5 168 16.5 4.84 9.03 1.81 98.0 19.1 91.5 33.1 33618.1 3.30 9.28 2.10 69.7 10.2 54.9 16.8 504 22.8 3.96 9.68 2.85 52.24.05 37.9 16.5 672 14.7 0.964 7.32 1.34 42.6 6.85 29.5 14.8 840 15.11.74 7.40 2.46 33.6 6.39 22.3 10.6 1008 14.7 3.47 6.82 2.06 28.5 6.2420.2 11.2 1176 13.2 2.99 5.96 1.76 24.1 7.04 16.6 9.37 1344 12.4 2.346.10 1.79 20.7 3.07 14.1 8.88 1512 12.0 0.917 5.81 1.81 19.7 5.98 13.47.16 1680 12.3 1.95 5.42 2.01 18.4 3.30 11.5 5.77 1848 10.6 0.83 5.181.51 14.3 1.35 11.4 6.39 2016 9.83 2.06 4.30 2.03 14.9 1.75 9.86 3.752184 10.2 2.42 4.55 1.36 12.6 0.755 8.87 3.37 2520 9.45 2.16 5.54 1.8211.6 2.06 9.27 3.53 2856 8.26 0.737 4.78 1.61 10.5 2.65 7.49 3.02 31926.82 1.38 4.04 1.02 8.67 2.71 6.28 2.52 3528 6.83 2.27 4.02 1.05 6.922.09 5.68 2.79 3864 6.69 0.794 3.95 0.866 5.90 2.21 5.00 2.53 4200 6.411.72 3.49 0.987 4.41 2.04 3.74 2.03 ≈ CV % 8-29 NC-47 6-46 26-63 T max(h) 24 24 18 120 83 Cmax 68.1 17.6 24.0 1.14 215 66.7 94.2 33.3 (ng/mL)T last (h) 4200 4200 4200 4200 AUClast 50200 4240 24800 5520 10900012300 75200 28700 (ng*h/mL) AUC₀₋₂₈₅₆ 41000 2880 19300 4150 99200 1320067600 26100 (ng*h/mL) AUCinf NC NC 121000 11400 85500 28800 (ng*h/mL)

Study in Rats: for Formulation 1A, i.e. the nano-suspension of 200 mg/mlconcentration, and dosed SC at 40 mg/kg (StDev=standard deviation) andIM at 40 mg/kg (in this case, small sample size applied to calculationof summary variable)

Plasma concentration of bedaquiline (BDQ) or its metabolite (M2) SC at40 mg/kg IM at 40 mg/kg Time (h) BDQ StDev M2 StDev BDQ StDev M2 StDev 142.1 11.4 BOL* NC 329 256 12.0 5.77 4 81.5 17.0 11.7 3.50 365 176 39.516.7 7 98.7 25.2 20.3 4.15 385 124 68.6 30.4 24 337 57.0 127 20.8 43641.2 217 36.3 168 92.4 33.6 100 17.7 94.9 25.1 89.9 21.8 336 62.7 5.6162.9 25.3 53.0 13.0 48.6 21.2 504 42.1 6.21 45.8 30.7 36.2 7.49 26.612.0 672 28.4 1.04 32.4 22.5 22.4 3.35 15.8 5.31 840 20.8 3.67 21.1 13.425.9 2.48 11.1 2.50 1008 16.5 4.56 16.2 12.0 12.0 1.55 8.13 1.82 117612.7 4.58 12.7 10.0 9.05 1.23 5.74 1.07 1344 12.0 7.04 10.1 9.35 7.330.739 4.20 1.18 1512 7.02 2.77 7.61 6.32 4.69 0.384 3.05 0.640 1680 6.052.79 6.02 NC 4.57 0.378 2.63 0.242 1848 4.95 2.56 5.35 5.22 4.05 0.1921.56 NC 2016 4.36 2.12 4.08 NC 3.21 0.646 1.92 0.246 2184 3.77 1.94 3.49NC 2.50 0.0231 1.80 0.102 2520 2.72 1.67 2.97 2.62 2.27 0.437 1.45 0.1212856 2.51 0.880 2.50 2.16 1.56 0.335 0.926 0.0759 3192 1.51 0.892 2.14NC 1.26 0.275 0.841 0.0826 3528 1.50 NC 1.28 NC 1.37 NC BOL* NC 38640.887 NC BOL* NC BOL* NC BOL* NC 4200 0.753 NC BOL* NC BOL* NC BOL* NC ≈CV % NC-61 NC-90 1-78 NC-48 T max (h) 24 24 16 24 Cmax 337 57.0 127 20.8505 96.6 217 36.3 (ng/mL) T last (h) 3900 580 3500 670 3500 340 3300 190AUClast 79100 3100 67200 33600 80400 8260 53400 10700 (ng*h/mL)AUC₀₋₂₈₅₆ 77300 4240 65400 31200 79400 8800 53000 10700 (ng*h/mL) AUCinf80100 2890 68600 34500 81200 8230 54000 10700 (ng*h/mL) NC = notcalculated BOL* = below limit of quantification (0.75 ng/mL or 1.5ng/mL)

Study in Dogs: for Formulation 1B, i.e. the micro-suspension of 200mg/ml concentration, and dosed SC at 40 mg/kg (StDev=standard deviation)and IM at 40 mg/kg.

Plasma concentration of bedaquiline (BDQ) or its metabolite (M2) SC at40 mg/kg IM at 40 mg/kg Time (h) BDQ StDev M2 StDev BDQ StDev M2 StDev 11.55 0.0794 BOL* NC 765 136 BOL* NC 4 5.43 1.48 BOL* NC 703 292 14.95.87 7 8.80 2.48 BOL* NC 735 274 21.2 8.72 24 21.9 13.0 BOL* NC 349 28.327.7 9.76 168 192 261 32.9 40.1 351 65.6 69.8 11.6 336 160 173 47.1 46.9355 30.2 94.2 7.02 504 149 151 50.4 51.5 338 30.0 93.8 13.2 672 123 10946.2 43.1 284 37.8 90.6 16.9 840 161 138 53.1 48.2 315 30.1 96.2 3.241008 125 104 50.1 40.1 227 25.2 80.7 4.11 1176 116 96.4 42.8 34.9 18745.1 60.8 13.0 1344 110 96.2 40.4 31.9 172 38.7 52.8 11.1 1512 108 93.541.0 35.7 171 34.4 62.1 14.2 1680 136 107 39.7 31.9 183 37.6 51.8 6.851848 93.0 75.4 37.4 30.4 135 43.4 46.9 9.40 2016 89.5 71.4 35.9 26.0 12131.1 41.2 7.86 2184 82.0 59.8 28.6 23.4 108 28.4 37.1 9.31 2520 83.158.8 29.8 24.3 88.0 28.4 29.2 7.60 2856 75.3 53.5 28.8 23.2 74.3 23.526.4 5.30 3192 60.3 36.1 23.8 15.4 58.7 17.6 22.1 5.17 3528 59.1 34.320.3 13.4 54.0 17.2 18.9 6.60 3864 52.8 26.0 20.1 11.0 45.4 15.8 16.45.17 4200 51.7 30.2 20.4 13.2 40.9 14.6 15.6 4.63 ≈ CV % 5-136 NC-1228-42 3-41 T max (h) 620 390 780 260 3.0 620 260 Cmax 219 237 55.3 47.7822 211 103 6.58 (ng/mL) T last (h) 4200 4200 4200 4200 AUClast 402000335000 138000 114000 652000 105000 193000 27900 (ng*h/mL) AUCinf NC NCNC 690000 NC NC NC (ng*h/mL) NC = not calculated BOL* = below limit ofquantification (3.75 ng/mL)

Study in Dogs: for Formulation 1A, i.e. the nano-suspension of 200 mg/mlconcentration, and dosed SC at 40 mg/kg (StDev=standard deviation) andIM at 40 mg/kg (in this case, small sample size applied to calculationof summary variable)

Plasma concentration of bedaquiline (BDQ) or its metabolite (M2) SC at40 mg/kg IM at 40 mg/kg Time (h) BDQ StDev M2 StDev BDQ StDev M2 StDev 1274 466 BOL* NC 4000 1510 11.1 1.35 4 194 197 BOL* NC 3570 620 41.7 7.257 157 134 4.94 NC 2690 842 51.5 10.6 24 167 52.6 8.68 6.84 742 82.3 62.617.2 168 692 217 127 54.6 568 142 108 27.1 336 386 55.4 132 29.0 41219.2 112 24.4 504 318 76.8 110 7.77 321 13.1 98.4 21.0 672 244 28.4 93.814.9 240 29.6 84.1 25.0 840 255 29.7 93.0 5.84 254 12.7 89.4 24.5 1008197 40.6 74.8 2.20 172 23.4 63.7 16.2 1176 158 21.4 59.5 4.92 149 10.058.7 13.0 1344 133 19.5 47.3 2.10 139 13.2 47.1 12.8 1512 124 25.4 46.45.82 120 8.62 41.6 10.6 1680 136 24.0 43.6 3.40 126 17.0 44.5 11.7 184889.6 23.8 33.8 3.53 95.2 2.14 32.7 4.60 2016 84.5 18.8 31.6 3.04 89.916.7 31.7 7.72 2184 80.5 25.4 27.9 3.52 78.4 5.66 24.5 6.07 2520 59.714.8 21.1 2.04 57.4 4.47 19.4 3.07 2856 53.9 18.0 19.6 4.25 54.8 3.0418.7 2.77 3192 45.2 16.7 16.4 4.19 42.9 4.32 14.5 2.80 3528 40.0 12.314.9 3.55 36.1 1.40 12.4 2.06 3864 34.5 13.1 12.8 3.20 32.3 2.42 11.51.82 4200 31.1 14.1 12.1 4.02 25.4 1.37 9.49 1.42 ≈ CV % 12-170 NC-792-38 12-30 T max (h) 168 280 97 3 2.0 280 97 Cmax 692 217 140 42.8 41501290 121 21.7 (ng/mL) T last (h) 4200 4200 4200 4200 AUClast 58000082500 186000 8740 641000 27700 174000 32900 (ng*h/mL) AUCinf NC NC215000 NC 677000 24800 193000 32200 (ng*h/mL) NC = not calculated BOL* =below limit of quantification (0.75 ng/mL)

Example 3 Evaluation of an Injectable, Long-Acting BedaquilineFormulation in the Paucibacillary Mouse Model of Latent TuberculosisInfection

The objective of this study was to use the paucibacillary mouse model oflatent tuberculosis infection (LTBI) to compare the bactericidalactivity of a long-acting bedaquiline (B_(LA)) formulation administeredevery 4 weeks for a total of 1, 2, or 3 doses to the activity of daily(5 days per week) oral dosing of B at the standard 25 mg/kg dose orlower doses matching to total drug doses administered as B_(LA). Theoriginal study scheme is presented in Table 1. The B_(LA) used for thisstudy is that described above in Example 1B, i.e. the microsuspension ata concentration of 200 mg/ml). The primary outcome was the decline inMycobacterium tuberculosis lung CFU counts during treatment.

TABLE 1 Original study scheme to evaluate the bactericidal activity ofB_(LA) in a mouse model of paucibacillary LTBI. Number of micesacrificed for lung CFU counts at the following time points: BCG M.tb.Treatment Total dose B LTBI treatment immunization challenge initiationDuring treatment Total over 12 weeks regimen* Week 12 Week 6 Day 0 Week4 Week 8 Week 12 mice (mg/kg) Untreated 5 5 5 5 5 5 30 na R₁₀ (5/7) 5 55 15 na P₁₅H₅₀ (1/7) 5 5 5 15 na B₂₅ (5/7) 5 5 5 15 1500  B₈ (5/7) 5 5 515 480 B_(5.33) (5/7) 5 5 5 15 320 B_(2.67) (5/7) 5 5 5 15 160B_(LA-160) (1/28) × 3 5  5 480 B_(LA-160) (1/28) × 2 5 5 10 320B_(LA-160) (1/28) × 1 5 5 5 15 160 Total mice 5 5 5 40  45  50  150 *R,rifampin; P, rifapentine; H, isoniazid; B, bedaquiline; B_(LA),long-acting bedaquiline formulation. All drug doses in mg/kg indicatedby subscript. Fractions in parentheses indicate dosing frequency, indays. B_(LA) is administered by intramuscular injection; all other drugsare administered by gavage. na, not applicable.

Justification of the Regimens

-   -   Untreated mice were used to determine the level and stability of        the paucibacillary infection.    -   R₁₀ (5/7) is an alternative regimen for treatment of LTBI in the        US and Canada, administered for 4 months. It was used here as a        control to qualify the model.    -   P₁₅H₅₀ (1/7) is an alternative regimen for treatment of LTBI in        the US, administered once weekly for 3 months (12 doses). It        proved at least as efficacious as 9 months of isoniazid. It is        the most intermittent of currently recommended regimens and        serves as a second control.    -   B₂₅ (5/7) is daily B at the human equivalent dose previously        studied in the paucibacillary model. It provides a total dose of        500 mg/kg every 28 days.    -   B₈ (5/7) is daily B at a dose that is reduced to provide the        same total dose (480 mg/kg) as the B_(LA) formulation dose        (i.e., 160 mg/kg) administered every 28 days×3 doses.    -   B_(5.33) (5/7) is daily B at a dose that is reduced to provide        the same total dose (320 mg/kg) as the B_(LA) formulation dose        (i.e., 160 mg/kg) administered every 28 days×2 doses.    -   B_(2.67) (5/7) is daily B at a dose that is reduced to provide        the same total dose (160 mg/kg) as the B_(LA) formulation dose        (i.e., 160 mg/kg) administered once.    -   B_(LA-160) (1/28)×3 is the B_(LA) formulation administered as        160 mg/kg every 28 days for a total of 3 doses. Thus, the total        B dose will match that of the B₈ (5/7) group at each 28-day        interval.    -   B_(LA-160) (1/28)×2 is the B_(LA) formulation administered as        160 mg/kg every 28 days for a total of 2 doses, beginning on        Day 0. Thus, the total B dose administered by Week 12 (320        mg/kg) will be the same as that of the B_(5.33) (5/7) group.    -   B_(LA-160) (1/28)×1 is the B_(LA) formulation administered as        160 mg/kg just once on Day 0. Thus, the total B dose        administered by Week 12 (160 mg/kg) will be the same as that in        the B_(2.67) (5/7) group.

Final Results

All CFU count data are finalized and presented below in Table 2. Due todelays in finalizing institutional agreements and obtaining the B_(LA)supply, treatment was not initiated until approximately 13 weeks afterthe M. tuberculosis challenge infection, and the time line in Table 2has been adjusted accordingly. For comparison between differenttreatment groups, statistical significance was assessed using one-wayANOVA adjusted with Bonferroni's multiple comparisons test.

TABLE 2 Final M. tuberculosis lung CFU count data. Mean (SD) log₁₀ M.tuberculosis CFU/lung at the following time points: Total dose BCG M.tb.Treatment B over 12 LTBI treatment immunization challenge initiationDuring treatment weeks regimen* Week 19 Week 13 Day 0 Week 4 Week 8 Week12 (mg/kg) Untreated na 2.11 (0.09) 4.75 (0.27) 4.71 (0.48) 4.60 (0.27)4.94 (0.29) na R₁₀ (5/7) 3.39 (0.46) 2.74 (0.62) 1.27 (0.85) na P₁₅H₅₀(1/7) 2.67 (0.25) 0.79 (0.80) 0.28 (0.41) na B₂₅ (5/7) 3.01 (0.45) 0.82(0.49) 0.07 (0.09) 1500  B₈ (5/7) 3.30 (0.12) 2.42 (0.26) 0.69 (0.43)480 B_(5.33) (5/7) 3.83 (0.25) 3.15 (0.47) 1.98 (0.17) 320 B_(2.67)(5/7) 3.96 (0.35) 3.52 (0.38) 3.16 (0.24) 160 B_(LA-160) (1/28) × 3 1.23(0.16) 480 B_(LA-160) (1/28) × 2 2.31 (0.40) 1.63 (0.40) 320 B_(LA-160)(1/28) × 1 3.55 (0.32) 3.31 (0.38) 1.83 (0.34) 160 *R, rifampin; P,rifapentine; H, isoniazid; B, bedaquiline, B_(LA), long-actingbedaquiline formulation. All drug doses in mg/kg indicated by subscript.Fractions in parentheses indicate dosing frequency, in days. SD,standard deviation. na, not applicable.

BCG immunization. One-hundred fifty female BALB/c mice were infected byaerosol with M. bovis rBCG30. A culture suspension with an OD₆₀₀ of 1.03was diluted 10-fold and then used for aerosol infection. Theconcentration of the bacterial suspension was 6.88 lop) CFU/mL, whichresulted in a mean implantation of 3.05 (SD 0.10) log₁₀ CFU/lung. Sixweeks later, at the time of the M. tuberculosis challenge infection, themean BCG burden in the mouse lungs was 4.95 (SD 0.11) log₁₀ CFU. By Day0, the BCG burden had decreased and stabilized at 3.27 (SD 0.45) log₁₀CFU/lung, with similar lung burdens observed in the untreated mice atWeeks 4, 8, and 12. Thus, a low-level, stable BCG infection wasestablished in the lungs of these mice as expected.

M. tuberculosis challenge. Six weeks after BCG immunization, mice wereinfected by aerosol with M. tuberculosis H37Rv. A culture suspensionwith an OD₆₀₀ of 0.850 was diluted ˜100-fold and then used for aerosolinfection. The concentration of the bacterial suspension was 4.73 log₁₀CFU/mL, which resulted in a mean implantation of 2.11 (SD 0.09) lop)CFU/lung. This implantation was approximately 1 log₁₀ CFU higher thanwas intended. By Day 0, the M. tuberculosis burden had stabilized ataround 4.8 log₁₀ CFU/lung, with similar lung burdens observed in theuntreated mice at Weeks 4, 8, and 12. Thus, despite the higherimplantation, a stable M. tuberculosis infection was established in thelungs of these mice, with the stabilized lung CFU burden correspondinglynearly 1 log₁₀ CFU higher than observed in previous experiments (1-3).

Assessment of bactericidal activity (Table 2). Compared to the M.tuberculosis CFU counts in the lungs of untreated mice, the R₁₀ (5/7)control regimen reduced the mean CFU count by approximately 1, 2 and 3log₁₀ CFU/lung after 4, 8, and 12 weeks of treatment, respectively. TheP₁₅H₅₀ (1/7) control regimen resulted in reductions of about 2, 3, and4.5 log₁₀ CFU after 4, 8, and 12 weeks of treatment, respectively. Therelative magnitudes of the decline in lung CFU counts for both controlregimens are as expected based on previous studies (1,2). B₂₅ (5/7)resulted in a reduction of about 1.7, 4.0, and 4.9 log₁₀ CFU/lung after4, 8, and 12 weeks of treatment, results which were also expected basedon previous studies (1-2). Thus, the higher implantation and Day 0 CFUcounts did not affect the relative activity of the drugs against thisstabilized bacterial population in the mouse lungs.

For all B test regimens, there was increasing activity with increasingdose observed at Weeks 4, 8, and 12. For mice that received one or twodoses of B_(LA-160) (1/28), the decrease in lung CFU counts relative tountreated mice was equivalent to the decrease in mice that received thesame total dose administered as a daily oral regimen, B₈ (5/7), for 4 or8 weeks, respectively (p>0.05 at both time points). One dose ofB_(LA-160), delivering 160 mg/kg at Day 0, resulted in a decline ofabout 1.3 log₁₀ CFU/lung, and four weeks of B₈ (5/7) resulted in adecline of about 1.5 log₁₀ CFU/lung. After two doses of B_(LA-160)(1/28) or 8 weeks of B₈ (5/7), the CFU counts in the lungs decreased byan additional 1 log₁₀ in mice that received either of these regimens.After 12 weeks of treatment, the CFU counts in the lungs were lower inmice that had received one dose of B_(LA-160) than in the mice thatreceived the same total dose of bedaquiline (160 mg/kg) via daily dosingwith B_(2.67) (5/7) (p=0.0002), with the former regimen resulting in adecline of about 3 log₁₀ CFU/lung and the latter resulting in a declineof 1.7 log₁₀ CFU/lung, compared to the lung counts in the untreatedcontrol mice. In mice that received a total bedaquiline dose of 320mg/kg, either through two doses of B_(LA-160) or through daily dosing ofB_(5.33) (5/7), the decline in lung CFU counts was the same at about 3log₁₀ CFU/lung (p>0.05). For mice that received a total bedaquiline doseof 480 mg/kg via three doses of B_(LA-160) (1/28), the lung CFU countswere higher than in mice that received the equivalent total dose throughdaily dosing with B₈ (5/7), although the difference was notstatistically significant.

After 12 weeks of treatment, nearly all test regimens had equivalentbactericidal activity as the R₁₀ (5/7) control regimen, with only theB_(2.67) (5/7) regimen being significantly less bactericidal than thiscontrol (p<0.0001). The test regimen B₈ (5/7) demonstrated equivalentbactericidal activity to both the P₁₅H₅₀ (1/7) and B₂₅ (5/7) controlregimens, while all other test regimens were significantly lessbactericidal than either of these control regimens at Week 12. However,CFU data recorded at the Week 12 time point may not reflect the overallefficacy of long-acting bedaquiline regimens. In mice that received asingle dose of B_(LA-160) on Day 0, bacterial killing was still observed12 weeks after administration. Thus, it is conceivable that thebacterial burden in the lungs of mice that received 2 and 3 doses ofB_(LA-160) would still further decrease for at least 12 weeks afteradministration of the last dose (if not longer). Also of interest isthat the single dose of B_(LA-160) seemed to exert greater bactericidalactivity from weeks 1 to 4 and from weeks 9 to 12, compared to weeks 5to 8 post-administration, suggesting the possibility of biphasic Brelease kinetics from the long-acting vehicle.

CONCLUSIONS

-   -   Despite a higher bacterial implantation than anticipated, a        stable M. tuberculosis infection was established in BALB/c mice        that was suitable for evaluation of LTBI treatment regimens.    -   After 12 weeks of treatment, once-monthly dosing with B_(LA-160)        demonstrated superior or equivalent bactericidal activity        compared to daily dosing for total bedaquiline doses of 160 or        320 and 480 mg/kg, respectively.    -   The bactericidal activity observed from a single dose of        B_(LA-160) was evident for at least 12 weeks after        administration, and likely CFU counts would continue to decrease        in the lungs of mice that received 2 and 3 doses. Taken together        with the higher-than-expected baseline bacterial burden in this        experiment, these findings suggest that cure after 2 or 3        injections may be possible. Thus, it will be critical to        evaluate the sterilizing activity of these B_(LA) regimens over        longer time periods to truly understand their potential for use        in LTBI treatment.

REFERENCES

-   1) Zhang, T., Li, S., Williams, K., Andries, K.,    Nuermberger, E. 2011. Short-course chemotherapy with TMC207 and    rifapentine in a murine model of latent tuberculosis infection. Am.    J Respir. Crit. Care Med. 184:732-737.-   2) Lanoix, J. P., Betoudji, F., Nuermberger, E. 2014. Novel regimens    identified in mice for treatment of latent tuberculosis infection in    contacts of multidrug-resistant tuberculosis cases. Antimicrob.    Agents Chemother. 58:2316-2321.-   3) Zhang, T., M. Zhang, I. M. Rosenthal, J. H. Grosset, and E. L.    Nuermberger. 2009. Short-course therapy with daily rifapentine in a    murine model of latent tuberculosis infection. Am. J Respir. Crit    Care Med. 180:1151-1157.

1-14. (canceled)
 15. A pharmaceutical composition for administration byintramuscular or subcutaneous injection, comprising a therapeuticallyeffective amount of bedaquiline, or a pharmaceutically acceptable saltthereof, in the form of a suspension of micro- or nanoparticlescomprising: (a) bedaquiline, or a pharmaceutically acceptable saltthereof, in micro- or nanoparticle form; and (b) a pharmaceuticallyacceptable aqueous carrier.
 16. The pharmaceutical composition accordingto claim 15, wherein bedaquiline is in its non-salt or free form or inthe form of a fumarate salt.
 17. The pharmaceutical compositionaccording to claim 15, wherein the average effective particle size ofthe bedaquiline, or a pharmaceutically acceptable salt thereof, micro-or nanoparticles is below about 50 μm.
 18. The pharmaceuticalcomposition according to claim 15, wherein the average effectiveparticle size of the bedaquiline, or a pharmaceutically acceptable saltthereof, micro- or nanoparticles is below about 200 nm.
 19. A method fortreating a subject infected with a pathogenic mycobacterial infectioncomprising administering to the subject, by intramuscular orsubcutaneous injection, a composition in the form of a suspensioncomprising: micro- or nanoparticles of bedaquiline, or apharmaceutically acceptable salt thereof, and a surface modifier; and apharmaceutical acceptable aqueous carrier, wherein the amount ofbedaquiline, or the pharmaceutically acceptable salt thereof, in thesuspension is effective in producing a minimum plasma level ofbedaquiline that is sufficient to treat the pathogenic mycobacterialinfection.
 20. The method of claim 19, wherein the pathogenicmycobacterial infection comprises Mycobacterium tuberculosis in alatent/dorman form or Mycobacterium leprae.
 21. The method of claim 19,wherein the suspension is administered intermittently at a time intervalof at least one week to two years.
 22. The method of claim 19, whereinthe suspension is administered once every two weeks, once every month,or once every three months.
 23. The method of claim 19, wherein theadministration is preceded with a lead-in treatment phase.
 24. Themethod of claim 23, wherein the lead-in treatment phase comprises anadministration course lasting for one week, two weeks, three weeks, orone month.
 25. The method of claim 19, wherein the minimum plasma levelof bedaquiline is above about 10 ng/ml.
 26. The method of claim 19,wherein the amount of bedaquiline, or the pharmaceutically acceptablesalt thereof is calculated on a basis of about 1 mg/day to about 150mg/day of bedaquiline.
 27. The method of claim 19, wherein bedaquilineis in its non-salt or free form or in the form of a fumarate salt. 28.The method of claim 19, wherein the average effective particle size ofthe bedaquiline, or the pharmaceutically acceptable salt thereof, micro-or nanoparticles is below about 50 μm.
 29. The method of claim 19,wherein the average effective particle size of the bedaquiline, or thepharmaceutically acceptable salt thereof, nanoparticles is below about200 nm.
 30. The method of claim 19, wherein the surface modifier isselected from the group of poloxamers, α-tocopherol polyethylene glycolsuccinates, polyoxyethylene sorbitan fatty acid esters, and salts fornegatively charged phospholipids.
 31. The method of claim 30, whereinthe surface modifier is selected from Pluronic™ F108, Vitamin E TGPS,Tween™ 80, and Lipoid™ EPG.
 32. The method of claim 19, wherein therelative amount (w/w) of bedaquiline to the surface modifier is in therange of 1:2 to about 20:1.
 33. The method of claim 19, wherein thecomposition comprises by weight based on the total volume of thecomposition: (a) from 10% to 70% (w/v) of bedaquiline (or apharmaceutically acceptable salt thereof but where the w/v is calculatedon the basis of its non-salt form); (b) from 0.5% to 20% (w/v) of awetting agent; (c) from 0% to 10% (w/v) of one or more buffering agents;(d) from 0% to 20% (w/v) of a isotonizing agent; (e) from 0% to 2% (w/v)preservatives; and (f) water for injection q.s. as 100%.
 34. The methodof claim 19, further comprising co-administering other anti-TB drugs.35. A pharmaceutical composition for administration by intramuscular orsubcutaneous injection, comprising a therapeutically effective amount ofbedaquiline, or a pharmaceutically acceptable salt thereof, in the formof a suspension of micro- or nanoparticles comprising: (a) bedaquiline,or a pharmaceutically acceptable salt thereof, in micro- or nanoparticleform, having a surface modifier absorbed to the surface thereof; and (b)a pharmaceutically acceptable aqueous carrier, wherein the bedaquiline,or a pharmaceutically acceptable salt thereof is suspended.